Biological Control of Plant Pathogenic Microorganisms

ABSTRACT

The present invention relates to the use of an isolated Aureobasidium pullulans yeast strain YBCA5 as a biological control agent. Processes and compositions for the biological control of phytopathogenic bacteria and fungi using YBCA5 are also provided.

FIELD OF THE INVENTION

This invention relates generally to methods of using yeasts for thebiological control of plant pathogenic bacteria and fungi. Inparticular, the invention relates to a novel yeast strain havingbiological control activity, and to methods of using this strain toinhibit the survival, growth and/or proliferation of plant pathogenicbacteria and fungi on fruit or vegetable plants.

BACKGROUND OF THE INVENTION

Plant disease represents a significant economic cost to modernagriculture. Current systems of agriculture often require one or a fewcrops or plant types to be grown over a large area. Such an ecologicallyunbalanced system is susceptible to disease.

Traditionally, control of disease causing plant pathogens such asbacteria and fungi has been carried out using chemical pesticides.However, the use of chemicals is subject to a number of disadvantages.Pathogens can and have developed tolerance to chemicals over time,producing increasingly pesticide resistant populations. Chemicalresidues may also pose environmental hazards as well as raising healthconcerns. In particular, consumers have become increasingly concernedabout chemical residues on plants and in food and wine, and theireffects on human health and the environment.

Biological control represents an alternative means of controlling plantdisease which reduces dependence on chemicals. Such “natural” methodsenjoy greater public acceptance, and may be more effective andsustainable than chemical control methods.

Pseudomonas is a genus of Gram-negative, aerobic gammaproteobacteria,belonging to the family Pseudomonadaceae. The genus contains 191 validlydescribed species, of which a number are plant pathogens. Within thegenus Pseudomonas spp., P. syringae is a prolific plant pathogen thatexists as over 50 different pathovars (pv.), many of which demonstrate ahigh degree of host-plant specificity. Numerous other Pseudomonasspecies can also act as plant pathogens, most notably all of the othermembers of the P. syringae subgroup. For example, commercially importantdiseases caused by P. syringae pathovars include bacterial blast ofstone fruits, bacterial speck of tomato, and blight in peas.

Pseudomonas syringae pv. actinidiae (Psa) is a serious bacterial diseaseaffecting kiwifruit. Psa was first recorded in New Zealand in earlyNovember 2010, and as of 18 Jul. 2013, 75% of hectares of kiwifruit wereon orchards with some Psa infection. The immediate cost of Psa to theNew Zealand kiwifruit industry is estimated to be between $310 millionand $410m from 2013 to 2018, and more than double that in the long-termfor lost development.

As with many bacterial plant diseases, control options are limited. Themain solutions currently in use are crop hygiene, chemical basedtreatment such as copper-based products, and/or plant defence elicitorssuch as acibenzolar-S-methyl (Actigard/Bion, Syngenta) and antibioticssuch as streptomycin sulphate and kasugamycin. However, severerestrictions have been placed on the use of these products and time ofthe growing season that they can be used (e.g. in New Zealand). Inaddition, the use of some of these products are prohibited in some keyexport regions e.g., Streptomycin is not permitted for use onhorticulture in Europe.

It has been estimated that in the 2012 season in New Zealand, $13Million was spent on sprays to protect kiwifruit against Psa. This isthe chemical cost alone. Other management costs have not been factoredinto this estimate. Outside of New Zealand, Psa is also a critical issuein Europe (Italy/France), South America and potentially in China andSouth Korea.

Botrytis cinerea and recently identified B. pseudocinerea arephytopathogenic fungi (telemorph Botryotinia fuckeliana) and are thecausal agents of the grey mold (Botrytis blight) disease. Some estimatesof global crop losses resulting from Botrytis spp. are on the order of10-100 billion Euros per year (http://www.genoscope.cns.fr). Botrytisspp. is also the causative agent of bunch rot of grapes, and isestimated to cause losses of $18 million dollars per annum to the NewZealand wine industry alone. Botrytis spp. control has been by way offungicides. As with the use of chemical treatments to control pathogenicbacteria, this practice is unsustainable because fungicide resistance iswidespread in many vineyards and there is consumer pressure forreduction in pesticide residue.

Brown rot on fruit is caused by Monilinia spp. fungi. Monilinia spp. arepathogens of many economically important crops in the Family Rosaceaeincluding cherries, plums, peaches, apricots, strawberries, raspberries,apples and pears. Monilinia spp. are also pathogens of many floweringplants within the Family Ericaceae. Damage caused by Monilinia spp. canoften cause major losses to crops and valuable ornamental flowers. Thegenus Monilinia contains about thirty described species.

Importantly, the revenues lost due to the impact of phytopathogenicfungi represent a mere fraction of the total economic impact of thesepathogens worldwide. As with Botrytis, control of Monilinia spp. andSclerotinia spp. on economically important crops has traditionally beenby way of fungicides. Some estimates consider that the cost of chemicalcontrol of Botrytis spp. alone can reach $780 million for just one cropwith disease on treated plants still resulting in significant productionloss (Genescope, 2002); (Laluk, Kristin and Tesfaye Mengiste; 2010 inArabidopsis Book 2010, Vol. 8).

Accordingly, for a number of economic, health and environmentalsustainability reasons as discussed above, the use of chemical basedtreatments, plant defence elicitors and antibiotics has limitations.Therefore, there is a need for new biological control solutions, whichdo not have similar cost, health or environmental issues to chemicalbased treatments in order to provide sustainable management of thesediseases.

It is an object of the invention to provide at least one yeastbiological control agent and/or a composition comprising at least oneyeast biological control agent and/or methods of using such an agentand/or such a composition for controlling Pseudomonas spp. bacteria onat least one plant or part thereof, preferably Pseudomonas syringae pv.actinidiae (Psa); and/or to at least to provide the public with a usefulchoice.

SUMMARY OF THE INVENTION

In one aspect the invention relates to isolated Aureobasidium pullulansyeast strain YBCA5 (CBS Accession # 141880).

In another aspect the invention relates to a composition comprisingisolated Aureobasidium pullulans yeast strain YBCA5 (CBS Accession #141880) and an agriculturally acceptable carrier.

In another aspect the invention relates to a composition consistingessentially of isolated Aureobasidium pullulans yeast strain YBCA5 (CBSAccession # 141880) and an agriculturally acceptable carrier.

In another aspect the invention relates to a method of controllingPseudomonas spp. bacteria on a plant or part thereof, the methodcomprising contacting the plant or part thereof with YBCA5, or acomposition comprising YBCA5.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for controlling Pseudomonas spp. bacteriaon a plant or part thereof.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for controlling Pseudomonasspp. bacteria on a plant or part thereof.

In another aspect the invention relates to a method for controlling P.syringae pv. actinidiae (Psa) on a kiwifruit plant or part thereof, themethod comprising contacting the kiwifruit plant or part thereof withYBCA5, or a composition comprising YBCA5.

In another aspect the invention relates to a method for increasing theyield of a kiwifruit plant infected, or susceptible to infection withPsa, the method comprising applying YBCA5 or a composition comprisingYBCA5 to the kiwifruit plant or part thereof, and growing the kiwifruitplant or part thereof.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for controlling Psa on a kiwifruit plant orpart thereof.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for increasing the yield of a kiwifruitplant infected, or susceptible to infection with Psa.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for controlling Psa on akiwifruit plant or part thereof.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for increasing the yield of akiwifruit plant infected, or susceptible to infection with Psa.

In another aspect the invention relates to a method of controlling atleast one phytopathogenic fungus on a plant or part thereof, the methodcomprising contacting the plant or part thereof with YBCA5, or acomposition comprising YBCA5.

In another aspect the invention relates to a method for increasing theyield of a fruit or vegetable plant infected with, or susceptible toinfection by a phytopathogenic fungus, the method comprising applyingYBCA5 or a composition comprising YBCA5 to the fruit or vegetable plantor part thereof YBCA5, and growing the plant or part thereof.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for controlling a phytopathogenic fungus ona fruit or vegetable plant or part thereof.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for increasing the yield of a fruit orvegetable plant or part thereof infected with, or susceptible toinfection by a phytopathogenic fungus.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for controlling at least onephytopathogenic fungus on a plant or part thereof.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for controlling at least onephytopathogenic fungus on a fruit or vegetable plant or part thereof.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for increasing the yield of afruit or vegetable plant susceptible to infection by at least onephytopathogenic fungus.

In another aspect the invention relates to at least one plant or partthereof treated with YBCA5, or a composition comprising YBCA5.

In another aspect the invention relates to at least one fruit orvegetable plant or part thereof treated with YBCA5, or a compositioncomprising YBCA5.

In another aspect the invention relates to at least one plant or partthereof treated with YBCA5, or a composition comprising YBCA5. In someembodiments the plant is a fruit or vegetable plant or part thereof. Inone embodiment the plant is a kiwifruit vine, a cherry tree or a grapevine.

While various embodiments of certain aspects of the invention are setout above, the invention is not limited thereto. Additional embodimentsof the aspects of the invention set out above are further described inthe Detailed Description and set out in the claims of the application.

Other aspects and embodiments of the invention may become apparent fromthe following description which is given by way of example only and withreference to the accompanying drawings.

It is intended that reference to a range of numbers disclosed herein(for example, 1 to 10) also incorporates reference to all rationalnumbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5,7, 8, 9 and 10) and also any range of rational numbers within that range(for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, allsub-ranges of all ranges expressly disclosed herein are hereby expresslydisclosed. These are only examples of what is specifically intended andall possible combinations of numerical values between the lowest valueand the highest value enumerated are to be considered to be expresslystated in this application in a similar manner.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of example only and withreference to the drawings in which:

FIG. 1. Psa severity (mean area of leaf necrosis) on potted ‘Hayward’seedlings treated with different concentrations of freshly fermentedYBCA5, compared with a water soluble granule formulation (YBCA5 granule)and inoculated with two doses (5×10⁶ per droplet and 2×10⁶ per 10 uldroplet) of Psa. Three 10 ul droplets of each dose of Psa were used perside of the leaf. Treatments were applied eight and one day (1d) priorto inoculation with Psa on 18 Sep. 2014 and assessed after 28 days.

FIG. 2. The effect of different isolates of Aureobasidium pullulans onthe severity of Psa leaf spot lesions on potted kiwifruit plants(‘Hayward’) compared to the untreated (Nil) in April 2016.

FIG. 3. Incidence of leaves with Psa necrosis on potted ‘Hayward’ plantsexposed to natural Psa inoculum at Te Puke Research Orchard and withtreatment applied on four occasions over a 30 day period. Leaf necrosisassessment was carried out 44 days after the first treatmentapplication.

FIG. 4. Field testing the efficacy of YBCA5. Nil is observed incidenceof leaf spotting on untreated control plants. Grower Std is the observedincidence of Psa leaf spotting on plants treated with Actigard andcopper. Low and high refer to the amount of YBCA4 and YBCA5 respectivelythat was applied to the plants. Field site was Maketu. The kiwifruitvariety was ‘Hayward’. All treatments were applied between bud burst andpre-flowering. A total of 5 spray treatments were carried out between 6and 12 days apart. For each of FIGS. 4-8, High rate is 2×10⁷ cells/mLand low rate is 1×10⁷.

FIG. 5. Field testing the efficacy of YBCA5. Nil is observed incidenceof defects on untreated control plants. Grower Std is the observedincidence of Psa leaf spotting on plants treated with Actigard andcopper. Low and high refer to the amount of YBCA4 and YBCA5 respectivelythat applied to the plants. Field site was Maketu. The kiwifruit varietywas ‘Hayward’. All treatments were applied between bud burst andpre-flowering. A total of 5 spray treatments were carried out between 6and 12 days apart.

FIG. 6. Field testing the efficacy of YBCA5 showing the mean severity ofleaf necrosis. Kiwifruit variety was ‘Hayward’. Grower std. iscopper+antibiotic. Treatments were applied from bud burst to firstflowering at two sites in Maketu. 6 treatments (sprays) were applied intotal, each 7-14 days apart.

FIG. 7. Field testing the efficacy of YBCA5 showing the mean severity ofbud browning. Kiwifruit variety was ‘Hayward’. Grower std. iscopper+antibiotic. Treatments were applied from bud burst to firstflowering at two sites in Maketu. 6 treatments (sprays) were applied intotal, each 7-14 days apart.

FIG. 8. Field testing the efficacy of YBCA5 showing the mean increase inyield (fresh weight/dry matter/fruit/m². Kiwifruit variety was‘Hayward’. Grower std. is copper+antibiotic. Treatments were appliedfrom bud burst to first flowering, once during flowering and once postfruit set. 7 treatments (sprays) were applied in total, each 7-14 daysapart. In each category: fresh weight, dry matter, fruit/m² (Gold3) andfruit/m² (‘Hayward’) the bars on the graph from left to right depict niltreatment, grower standard treatment (copper and antibiotic) and YBCA5.

FIG. 9. The effect of YBCA5 on the incidence of Monilinia fruit rot ofcherries (‘Sweet Valentine’) compared to the fungicide iprodione(Rovral® Aquaflo) in a lab based assay (Assay 1) in January-February2016.

FIG. 10. The effect of YBCA5 on the incidence of Botrytis spp. fruit rotof cherries (‘Sweet Valentine’) compared to the fungicide iprodione in alab based assay (Assay 2) in January-February 2016.

FIG. 11. The effect of YBCA5 on the incidence of Monilinia fruit rot ofcherries (‘Sweet Valentine’) compared to the fungicide captan in a labbased assay (Assay 3) in February-March 2016.

FIG. 12. The effect of YBCA5 on the incidence of Botrytis spp. fruit rotof cherries (‘Sweet Valentine’) compared to the fungicide captan in alab based assay (Assay 2) in February-March 2016.

FIG. 13. The effect of YBCA5 on the severity of Botrytis spp. rot oftable grapes (‘Autumn King’) compared to the fungicide captan in a labbased assay (Assay 5) in October-November 2015. Data is the mean of twoBotrytis spp. isolates.

FIG. 14. The effect of YBCA5 on the severity of kiwifruit rot due tophytopathogenic fungal infection post-harvest. Lesion size (mm) ofwounded ‘Hongyang’ kiwifruit after inoculation with Alternaria spp,Botrytis spp., Colletotrichum spp., Penicillium spp. or Phomopsis spp.and 6 days incubation. LSD (5%)=3.482, P fr=<.001.

FIG. 15. The effect of YBCA5 on the severity of kiwifruit rot due tophytopathogenic fungal infection post-harvest. Lesion size (mm) ofwounded ‘Hongyang’ kiwifruit after inoculation with Cryptosporiopsisspp. and 7 days incubation. LSD (5%)=1.945, P fr=<.001.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following definitions are presented to better define the presentinvention and as a guide for those of ordinary skill in the art in thepractice of the present invention.

Unless otherwise specified, all technical and scientific terms usedherein are to be understood as having the same meanings as is understoodby one of ordinary skill in the relevant art to which this disclosurepertains.

Examples of definitions of common terms in botany, microbiology,molecular biology and biochemistry can be found in Biology of Plants,Raven et al. (eds.), W. H. Freeman and Company, (2005); PlantPhysiology, Taiz et al. (eds.), Sinauer Associates, Incorporated,(2010); Botany: An Introduction to Plant Biology, J. D. Mauseth, Jones &Bartlett Learning, (2003); Methods for General and MolecularMicrobiology, 3rd Edition, C. A. Reddy, et al. (eds.), ASM Press,(2008); Encyclopedia of Microbiology, 2nd ed., Joshua Lederburg, (ed.),Academic Press, (2000); Microbiology By Cliffs Notes, I. Edward Alcamo,Wiley, (1996); Dictionary of Microbiology and Molecular Biology,Singleton et al. (2d ed.) (1994); Biology of Microorganisms 11^(th) ed.,Brock et al., Pearson Prentice Hall, (2006); Biodiversity of Fungi:Inventory and Monitoring Methods, Mueller et al., Academic Press,(2004); Genes IX, Benjamin Lewin, Jones & Bartlett Publishing, (2007);The Encyclopedia of Molecular Biology, Kendrew et al. (eds.), BlackwellScience Ltd., (1994); and Molecular Biology and Biotechnology: aComprehensive Desk Reference, Robert A. Meyers (ed.), VCH Publishers,Inc., (1995).

It is also believed that practice of the present invention can beperformed using standard botanical, microbiological, molecular biologyand biochemistry protocols and procedures as known in the art, and asdescribed, for example in Environmental Microbiology: Methods andProtocols, J. F. T. Spencer et al., Humana Press, (2004); EnvironmentalMicrobiology, P. D. Sharma, Alpha Science International, (2005);Environmental Microbiology, J. R. Leadbetter, Gulf ProfessionalPublishing, (2005) and other commonly available reference materialsrelevant in the art to which this disclosure pertains, and which are allincorporated by reference herein in their entireties.

The term “plant” as used herein encompasses whole plants and all partsof a plant from all stages of a plant lifecycle including but notlimited to vegetative and reproductive cells and tissues, propagules,seeds, embryos, fruits, shoots, stems, leaves, leaf sheaths and blades,inflorescences, roots, anthers, ligules, palisade, mesophyll, epidermis,auricles, palea, lemma and tillers.

The term “kiwifruit” is used herein as the common name for allcommercially grown fruit from the genus Actinidia. The most commonkiwifruit is the green-fleshed kiwifruit, from the species Actinidiachinensis var. deliciosa. Other species that are commonly eaten includegolden kiwifruit (A. chinensis var. chinensis), Chinese egg gooseberry(A. coriacea), baby kiwifruit (A. arguta), Arctic kiwifruit (A.kolomikta), red kiwifruit (A. melanandra; A. chinensis var. chinensis,),silver vine (A. polygama), and purple kiwifruit (A. purpurea).

The term “biological control agent” as used herein refers to agentswhich act as an antagonist of one or more plant pathogens. Antagonistsmay take a number of forms. In one form, the biological control agentmay out-compete the pathogen for available nutrients and/or space of thehost plant. In another form the biological control agent may render theenvironment unfavourable for the pathogen. Accordingly, the antagonistmechanisms include but are not limited to antibiosis, mycoparasitism,nutrient competition and physical displacement.

The terms “control”, “controlling”, “biocontrol” or “biological control”are used interchangeably herein to refer to the reduction of the amountof inoculum or disease-producing activity of a pathogen accomplished byor through one or more microorganisms. Generally comprehended is theprevention or reduction of infection by plant pathogenic bacteria orfungi, particularly plant pathogenic Pseudomonas spp., Botrytis spp.,Alternaria spp., Colletotrichum spp., Penicillium spp., Phomopsis spp.,Cryptosporiopsis spp., Monilinia spp., and Sclerotinia spp.,particularly or inhibition of the rate or extent of such infection,including any reduction in the survival, growth and/or proliferation ofthe bacteria or fungi. Curative treatment is also contemplated.

The term “statistically significant” as used herein refers to thelikelihood that a result or relationship is caused by something otherthan random chance. A result may be found to be statisticallysignificant using statistical hypothesis testing as known and used inthe art. Statistical hypothesis testing provides a “P-value” as known inthe art, which represents the probability that the measured result isdue to random chance alone. It is believed to be generally accepted inthe art that levels of significance of 5% (0.05) or lower are consideredto be statistically significant.

The term “effective amount” as used herein means an amount effective toprotect against, delay, reduce, stabilise, improve or treat plantpathogenic bacterial or fungal infection in and/or on a plant.

The terms “increasing the yield of a fruit or vegetable plant” and“increasing the yield of a kiwifruit plant” as used herein generallycomprehends increasing the rate of production of harvestable fruitand/or kiwifruit, the total number of harvestable fruit and/or kiwifruit(including due to absolute increase in fruit and/or kiwifruit numbers orreduction in disease symptoms leading to increased numbers of saleablefruits), and any increase in size of individual fruits and/or kiwifruitsproduced on a fruit or vegetable plant or kiwifruit plant treatedaccording to the invention. Increase is generally determined as comparedto an equivalent plant that is untreated with the strain or thecomposition of the invention.

An “agriculturally acceptable adjuvant” as used herein refers to acompound or material that is generally comprehended in the art ofagriculture as a useful additive in agricultural formulations or carriedout with agricultural treatments.

An “additional active agent” as used herein means any compound ormaterial that is capable of contributing to the control (as definedherein) of plant pathogenic Pseudomonas spp. bacteria or phytopathogenicfungi Botrytis spp., Alternaria spp., Colletotrichum spp., Penicilliumspp., Phomopsis spp., Cryptosporiopsis spp., Monilinia spp., andSclerotinia spp. by the yeasts useful in the present invention, or thatis capable of potentiating the effects of the yeasts useful in thisinvention in controlling plant disease caused by plant pathogenicbacteria and fungi

A “formulation agent” as used herein refers to any compound or materialthat facilitates or optimizes the production, handling, storage,transport, application and/or persistence of the composition of, or foruse in the invention on plants (as defined herein), but not limitedthereto.

An “agriculturally acceptable carrier” is used herein as is generallycomprehended in the art. A preferred agriculturally acceptable carrieris water, but not limited thereto.

The term “comprising” as used in this specification means “consisting atleast in part of”. When interpreting each statement in thisspecification that includes the term “comprising”, features other thanthat or those prefaced by the term may also be present. Related termssuch as “comprise” and “comprises” are to be interpreted in the samemanner.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to a novel Aureobasidiumpullulans yeast strain YBCA5 and to compositions comprising YBCA5 and anagriculturally acceptable carrier. In some embodiments the compositionsalso comprise an agriculturally acceptable adjuvant. The novel strainand compositions of the invention are useful for the biocontrol of plantdisease caused by plant pathogenic bacteria and phytopathogenic fungi,particularly Pseudomonas spp. bacteria and Botrytis spp., Sclerotiniaspp., Alternaria spp., Colletotrichum spp., Penicillium spp., Phomopsisspp., Cryptosporiopsis spp., and Monilinia spp. fungi. The inventionalso relates to methods of controlling phytopathogenic bacteria and/orfungi selected from the group consisting of Pseudomonas spp. bacteria,Botrytis spp., Sclerotinia spp., Penicillium spp., Colletotrichum spp.,Alternaria spp., Phomopsis spp., Cryptosporiopsis spp., and Moniliniaspp. fungi on a plant or part thereof by contacting the plant or partthereof with YBCA5.

The applicants are the first to provide the isolated yeast strain YBCA5,and compositions comprising YBCA5 and an agriculturally acceptablecarrier that are effective at controlling Pseudomonas spp. bacteria andphytopathogenic fungi on plants. In some embodiments YBCA5 or thecomposition comprising YBCA5 may also be formulated with anagriculturally acceptable adjuvant. The applicants are also the first toprovide methods of using the yeast, A. pullulans for biological controlof Pseudomonas spp. bacteria. In particular, the applicants are thefirst to show that a strain of A. pullulans yeast, or a compositioncomprising a strain of A. pullulans yeast is effective at inhibiting thesurvival, growth and/or proliferation of Pseudomonas syringae pv.actinidiae (Psa) on fruit or vegetable plants, particularly fruit orvegetable vines, particularly kiwifruit vines.

Without wishing to be bound by theory the applicants believe that theefficacy of the yeast strain and compositions of the invention relatesto either the ability of the yeast strain to competitively exclude Psaand/or phytopathogenic fungi, by excretion of an anti-microbial compoundor compounds, or by elicitation of plant defence mechanisms, or acombination of the above. Irrespective of the particular mode of action,the inventors have surprisingly found that YBCA5 is efficacious fortreating Psa disease on kiwifruit vines, for treating Botrytis spp. andMonilinia spp. infection on cherries and grapes, and for treatingAlternaria spp., Colletotrichum spp., Penicillium spp., Phomopsis spp.,Cryptosporiopsis spp, on apples and kiwifruit. YBCA5 is a particularlyeffective biological control agent against Pseudomonas spp. bacteria andphytopathogenic fungi. YBCA5 demonstrates the ability to surviveformulation and application protocols, rapidly colonise treated plants,and suppress growth of Pseudomonas spp. bacteria and of phytopathogenicfungi on treated plants and parts thereof. YBCA5 has been found to beparticularly effective at controlling P. syringae bacteria, particularlyP. syringae pv. actinidiae (Psa) bacteria, on kiwifruit vines, and atreducing and/or controlling, to varying degrees, post-harvest fruit rotdue to Botrytis spp., Sclerotinia spp., Penicillium spp., Colletotrichumspp., Alternaria spp., Phomopsis spp., Cryptosporiopsis spp, andMonilinia spp.

YBCA5 and Compositions

Accordingly, in one aspect the invention relates to isolatedAureobasidium pullulans yeast strain YBCA5 (CBS Accession # 141880).

The particular isolated A. pullulans strain YBCA5 of the invention wasdeposited on 26 Sep. 2016 for the purpose of patent procedure under theBudapest Treaty at Centraalbureau voor Schimmelcultures (CBS),Uppsalalaan 8,3584, CT Utrecht, The Netherlands. This isolate has beenaccorded deposit number CBS Accession # 141880.

The isolated A. pullulans yeast strain YBCA5 is a unicellular fungi ofthe Order Dothideales, Family Aureobasidiaceae, and genus Aureobasidum.Cells display a wide range of morphological variability. A. pullulanscultivated on potato dextrose agar produces smooth, faint pink,yeast-like colonies. Older colonies can be somewhat darker due to theproduction of chlamydospores. Primary conidia of A. pullulans are singlecelled, hyaline, smooth, ellipsoidal, and variable in shape and size. A.pullulans conidiophores are undifferentiated, intercalary or terminal,or arising as short lateral branches. Endoconidia are produced by A.pullulans intercalary cells. Hyphae are thin-walled, hyaline and smooth,with transverse septa. Growth occurs at 10-35° C. with optimal growthbeing 22-25° C.

In another aspect the invention relates to a composition comprisingYBCA5 (CBS Accession # 141880) and an agriculturally acceptable carrier.

In another aspect the invention relates to a composition consistingessentially of YBCA5 (CBS Accession # 141880) and an agriculturallyacceptable carrier.

In one embodiment the agriculturally acceptable carrier is water.

Again, without wishing to be bound by theory, the inventors believe thatthe when used as a biological control agent, YBCA5 must be in areproductively viable form. For most purposes YBCA5 desirablyincorporated into a composition in the form of reproductively viablecells. Preferably YBCA5 is incorporated into the composition as driedcells.

The concentration of cells in a composition of the invention will dependon the utility to which the composition is put. Optimizing theconcentration of cells for a particular application is believed to bewithin the skill in the art.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention ranges from about 1×10³ to about 1×10¹⁴,preferably about 1×10⁵ to about 1×10¹¹, preferably about 1×10⁶ to about1×10⁹, preferably about 1×10⁷ to about 1×10⁸, preferably about 2×10⁷ toabout 2×10⁸ CFU, preferably about 2×10⁹ to about 2×10¹⁰ CFU per gram forsolid compositions, and about 1×10⁷ to about 1×10⁸ CFU per millilitrefor liquid compositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention ranges from 1×10³ to about 1×10¹⁴,preferably 1×10⁵ to about 1×10¹¹, preferably from 1×10⁶ to about 1×10⁹,preferably 1×10⁷ to about 1×10⁸, preferably from 2×10⁷ to about 2×10⁸CFU, preferably from 2×10⁹ to about 2×10¹⁰ CFU per gram for solidcompositions, and from 1×10⁷ to about 1×10⁸ CFU per millilitre forliquid compositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention ranges from about 1×10³ to 1×10¹⁴,preferably about 1×10⁵ to 1×10¹¹, preferably about 1×10⁶ to 1×10⁹,preferably about 1×10⁷ to 1×10⁸, preferably about 2×10⁷ to 2×10⁸ CFU,preferably about 2×10⁹ to 2×10¹⁰ CFU per gram for solid compositions,and about 1×10⁷ to 1×10⁸ CFU per millilitre for liquid compositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention ranges from 1×10³ to 1×10¹⁴, preferably1×10⁵ to 1×10¹¹, preferably 1×10⁶ to 1×10⁹, preferably 1×10⁷ to 1×10⁸,preferably 2×10⁷ to 2×10⁸ CFU, preferably 2×10⁹ to 2×10¹⁰ CFU per gramfor solid compositions, and 1×10⁷ to 1×10⁸ CFU per millilitre for liquidcompositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention is about 2×10¹⁰ CFU per gram for solidcompositions, and about 2×10⁷ CFU per millilitre for liquidcompositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention is at least 2×10¹⁰ CFU per gram for solidcompositions, and at least 2×10⁷ CFU per millilitre for liquidcompositions, preferably the concentration of YBCA5 viable cells in acomposition of the invention is 2×10¹⁰ CFU per gram for solidcompositions, and 2×10⁷ CFU per millilitre for liquid compositions.

The composition of the invention may comprise or consist essentially ofYBCA5.

Concentrations of YBCA5 that are effective as a biological control agentin the composition of the invention may vary depending on the form theyeast is used in, physiological condition of the plant; type,concentration and degree of pathogen infection; temperature; season;humidity; soil type; stage in the growing season; age of the plant;number and type of conventional pesticides and fungicides being appliedand plant treatments (such as pruning, but not limited thereto). Allfactors may be taken into account in formulating YBCA5 in thecomposition of the invention or in a composition for use in a method ofthe invention.

YBCA5 may be prepared for use in the invention using standard liquidfermentation techniques known in the art and as described in theexamples herein. Growth is commonly effected under aerobic conditions ina bioreactor at suitable temperatures and pH for growth. Typical growthtemperatures are from 10 to 30° C., preferably 15 to 28° C., preferably25° C. Yeasts with optimal growth temperatures in the range of about36-38° C. are not preferred for use due to the potential for humanhealth risk. The pH of the growth medium is usually slightly acidic toneutral at pH 4.0 to 7.0, preferably 6.0.

Growth medium may be any known art medium suitable for culture ofAureobasidium species. In one embodiment the growth medium is potatodextrose agarose (PDA).

Other suitable growth media include Malt Yeast Extract Agar; aproprietary liquid broth culture media comprising molasses and urea; anda proprietary liquid growth media comprising sugar, urea, yeast extractand mono ammonium phosphate (MAP).

The cells of YBCA5 may be harvested using conventional filtering orsedimentary techniques such as centrifugation, or may be harvested dryusing continuous centrifugation. Cells can be used immediately or storedunder chilled conditions (1° C. to 7° C., preferably 2° C.), or may bedried. Preferably, cells are dried and formulated as dry yeast granules.For example, cells may be dried using a fluidized bed drier, but notlimited thereto. Preferably the dry yeast granules comprise at least 90%solids, preferably at least 95% solids, preferably about 96% solids.Preferably cells have a shelf life of at least two years. In oneembodiment shelf life is at least six months, preferably at least oneyear, preferably at least two years wherein the cells are maintainedunder chilled conditions. Preferably chilled conditions are 10° C. orless, but greater than 0° C. Preferably chilled conditions are selectedfrom the group consisting of 1° C., 2° C., 3° C., 4° C., 5° C., 6° C.,7° C., 8° C., 9° C. and 10° C. or variations within such temperaturesfrom about 1° C. to about 10° C.

In one embodiment the composition comprises an agriculturally acceptableadjuvant. In one embodiment the agriculturally acceptable adjuvant isselected from the group consisting of an additional active agent and aformulation agent.

In one embodiment the agriculturally acceptable adjuvant is one or moreadditional active agents. In one embodiment the agriculturallyacceptable adjuvant is one or more formulation agents.

In one embodiment the composition comprises a combination of one or moreadditional active agents and one or more formulation agents. In someembodiments the composition is formulated as pre-prepared composition orin a concentrated form. In some embodiments the composition comprises asolid or a liquid formulation.

In one embodiment composition of the invention comprises one or moreagriculturally acceptable adjuvants. In one embodiment theagriculturally acceptable adjuvants are selected from the group ofadditional active agents and formulation agents. Preferably the one ormore agriculturally acceptable adjuvant is an additional active agent.Preferably the one or more agriculturally acceptable adjuvant is aformulation agent.

In one embodiment the composition of the invention comprises acombination of one or more additional active agents and one or moreformulation agents.

In some cases it may also be desirable to include one or more additionalactive agents in the compositions of the invention where such additionalactive agents are capable of contributing to the control (e.g.,treatment and/or prevention) of plant pathogenic Pseudomonas spp.bacteria or plant pathogenic fungi including Botrytis spp., Sclerotiniaspp., Penicillium spp., Colletotrichum spp., Alternaria spp., Phomopsisspp., Cryptosporiopsis spp., and Monilinia spp., but not limitedthereto.

Suitable additional active agents for use in the present invention maybe capable of controlling Pseudomonas spp., particularly Psa directly,or plant pathogenic fungi including Botrytis spp., Sclerotinia spp.,Penicillium spp., Colletotrichum spp., Alternaria spp., Phomopsis spp.,Cryptosporiopsis spp. and Monilinia spp. (but not limited thereto), ormay be capable of potentiating the biocontrol effect of YBCA5 forcontrolling Pseudomonas spp., particularly Psa. Additional active agentsmay be included directly in the composition of or useful in theinvention, or may be applied separately, either simultaneously orsequentially as appropriate according to a method of the invention.

Suitable additional active agents include, but are not limited to plantdefence elicitors including acibenzolar-S-methyl (Actigard/Bion,Syngenta), Azelaic acid, Pipecolinic acid, Jasmonic acid, Seaweed Mix,Lema oil, Foodcoat (DOMCA), Fungicover (bioDURACAL agricultura) andIbuprofen, antagonistic microorganisms, inorganic salts includingcalcium, potassium or sodium salts, stimulating agents including uronicacids, amnnans, and β 1-3 glucans, antibiotics, and other antibacterialand antifungal compounds including small organic and inorganicmolecules.

By way of non-limiting example, one additional active agent that may beincluded in the composition of or for use in the invention is the plantdefence elicitor acibenzolar-S-methyl (Actigard/Bion, Syngenta).Actigard is a plant activator with a unique mode of action whichstimulates the natural systemic acquired resistance response found inmost plant species. Applied via foliar application, Actigard has nodirect activity against target pathogens, but helps to reduce Psasymptoms in Kiwifruit by inducing host plant resistance. Actigard is acomposition comprising 500 g/kg acibenzolar-S-methyl in the form of awater dispersible granule.

In one embodiment the composition of the invention comprises one or moreformulation agents.

In one embodiment the composition of the invention comprises acombination of one or more additional active agents and one or moreformulation agents.

In one embodiment, the composition of the invention is formulated as asolid or a liquid formulation.

In one embodiment the composition of the invention may comprise one ormore solid or liquid formulation agents. Any suitable formulationagent(s) may be used as known in the art. It is believed that theselection of a suitable formulation agent is within the skill of thosein the art. For example, a suitable formulation agent may be a compoundor other material that facilitates or optimizes the production,handling, storage, transport, application and/or persistence of thecomposition of, or for use in the invention on plants or on partsthereof, but not limited thereto.

Formulation agents can be specifically adapted for particular uses suchas, but not limited to, preservation and maintenance of the biologicalcontrol activity of the yeasts comprised in the composition of or foruse in the invention during transportation from production facilities,storage on site, or during preparation of a final treatment mixture.Formulation agents may also be used for other purposes such asfacilitating adhesion and persistence on plants or penetration intoplant tissues, but not limited thereto. A suitable formulation may besolid, liquid, alone or in combination. Particularly suitableformulation agents include surfactants, dispersants, preservatives,wetting agents, emulsifiers, humectants, stickers, spreaders,stabilizers, penetrants, adhesion agents, pH buffers, and nutrients,either alone or in various combinations as may be determined by theskilled worker.

The composition of the invention may be provided as a pre-preparedcomposition ready for use, or in a concentrated, solid or liquid form.

In one embodiment, the composition is a pre-prepared composition havinga solid or liquid formulation. In one embodiment the pre-preparedcomposition is a solid formulation selected from powders, pellets,granules and prills. In one embodiment the pre-prepared composition is aliquid formulation.

The composition of or for use in the invention may be provided in apre-prepared form, or in a concentrated form. If provided in a dry form,the pre-prepared composition may be provided as a powder, granule,pellet or prill, but not limited thereto. In the case of a dry form,YBCA5 in the composition is preferably in dehydrated, dried and/orencapsulated form. In some embodiments, the dehydrated, dried and/orencapsulated forms include additional protective agents as known in theart; e.g., lyoprotectants and the like.

In one embodiment, YBCA5 may be provided in granule form. For example,YBCA5 may be provided in a granule having at least 0.5×10¹⁰ CFU/gm,preferably 1×10¹⁰ CFU/gm, preferably 2×10¹⁰ CFU/gm. Where thepre-prepared composition is provided in a liquid form, particularly anaqueous form the composition may be provided as a dispersion, asuspension, a slurry, a cream, a paste or a gel, but not limitedthereto. Preferably the pre-prepared form is provided as an aqueousliquid form that is suitable for and/or is adapted for spraying. In oneembodiment a pre-prepared liquid form can be used per se for example asa dip to inoculate fruits, vegetables, seeds or plants, including plantcuttings.

In the pre-prepared composition of the invention, YBCA5 is formulatedfor use on plants, particularly kiwifruit vines. For example, the yeastsare mixed with an agriculturally acceptable carrier liquid that enablesspray applications, a fertilizer, an elicitor, an adjuvant, a wettingagent, or any other suitable additional agent as required. In thepre-prepared composition for use according to the methods of theinvention, YBCA5 may also be mixed with an agriculturally acceptablecarrier liquid that enables spray applications, a fertilizer, anelicitor, an adjuvant, a wetting agent, or any other suitable additionalagent as required.

The formulation of YBCA5 into a pre-prepared composition of theinvention and the final form of the pre-prepared composition forapplication to the plant or part thereof is believed to be within theskill in the art. For example, the final form of the composition isformulated with an agriculturally acceptable carrier such as water toform a spray, foam, drench, injectable, gel, dip or paste, but notlimited thereto. In one embodiment, a composition of the invention maybe applied to plants or parts thereof by spraying, dipping, rubbing orbrushing, or a combination thereof. Preferably the composition isformulated as an aqueous suspension or dispersion for spray or mistapplication to kiwifruit vines, cherry trees and/or fruit and grapevines and/or fruit and/or vegetables.

In one embodiment the composition of the invention is in concentratedform. In one embodiment the concentrated form is a solid form selectedfrom cakes, powders, granules, pellets and prills. In one embodiment theconcentrated form is a liquid formulation.

Where the composition of the invention is provided in a concentratedform it may require additional formulation by the user to produce acomposition ready for application to a plant or part thereof. Forexample, the concentrated form can be mixed with various formulationagents to form a final composition for plant application. A preferredformulation is agent is water or an aqueous solution in which anappropriate amount of the concentrated from of the composition isdissolved (e.g., granules or powders) or diluted (e.g., liquidsuspensions or dispersions) to obtain a final composition forapplication to a plant.

If the YBCA5 is dehydrated in the concentrated form then rehydration asknown in the art will be required if the composition for application tothe plant is intended to be in liquid form. Rehydration may be carriedout using customary precautions for rehydrating the yeast as known inthe art; for example rehydration may be achieved advantageously attemperatures between 20 and 25° C., but not limited thereto.

Methods—Pseudomonas spp.

In another aspect the invention relates to a method of controllingPseudomonas spp. bacteria on a plant or part thereof, the methodcomprising contacting the plant or part thereof with YBCA5, or acomposition comprising YBCA5.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for controlling Pseudomonas spp. bacteriaon a plant or part thereof.

In one embodiment the method or use comprises contacting the plant orpart thereof with reproductively viable cells of YBCA5.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention ranges from about 1×10³ to about 1×10¹⁴,preferably about 1×10⁵ to about 1×10¹¹, preferably about 1×10⁶ to about1×10⁹, preferably about 1×10⁷ to about 1×10⁸, preferably about 2×10⁷ toabout 2×10⁸ CFU, preferably about 2×10⁹ to about 2×10¹⁰ CFU per gram forsolid compositions, and about 1×10⁷ to about 1×10⁸ CFU per millilitrefor liquid compositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention ranges from 1×10³ to about 1×10¹⁴,preferably 1×10⁵ to about 1×10¹¹, preferably from 1×10⁶ to about 1×10⁹,preferably 1×10⁷ to about 1×10⁸, preferably from 2×10⁷ to about 2×10⁸CFU, preferably from 2×10⁹ to about 2×10¹⁰ CFU per gram for solidcompositions, and from 1×10⁷ to about 1×10⁸ CFU per millilitre forliquid compositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention ranges from about 1×10³ to 1×10¹⁴,preferably about 1×10⁵ to 1×10¹¹, preferably about 1×10⁶ to 1×10⁹,preferably about 1×10⁷ to 1×10⁸, preferably about 2×10⁷ to 2×10⁸ CFU,preferably about 2×10⁹ to 2×10¹⁰ CFU per gram for solid compositions,and about 1×10⁷ to 1×10⁸ CFU per millilitre for liquid compositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention ranges from 1×10³ to 1×10¹⁴, preferably1×10⁵ to 1×10¹¹, preferably 1×10⁶ to 1×10⁹, preferably 1×10⁷ to 1×10⁸,preferably 2×10⁷ to 2×10⁸ CFU, preferably 2×10⁹ to 2×10¹⁰ CFU per gramfor solid compositions, and 1×10⁷ to 1×10⁸ CFU per millilitre for liquidcompositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention is about 2×10¹⁰ CFU per gram for solidcompositions, and about 2×10⁷ CFU per millilitre for liquidcompositions.

In some embodiments the concentration of YBCA5 viable cells in acomposition of the invention is at least 2×10¹⁰ CFU per gram for solidcompositions, and at least 2×10⁷ CFU per millilitre for liquidcompositions, preferably the concentration of YBCA5 viable cells in acomposition of the invention is 2×10¹⁰ CFU per gram for solidcompositions, and 2×10⁷ CFU per millilitre for liquid compositions.

In one embodiment the at least one strain of Pseudomonas spp. isselected from the group consisting of P. syringae, P. amygdalia, P.avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P.ficuserectae, P. helianthi, P. lemiae, P. savastanoi, and P.viridiflava, or a pathovar thereof, or combinations thereof. Preferablythe at least one strain is P. syringae or a pathovar thereof, morepreferably the at least one strain is P. syringae pv. actinidiae (Psa).

In one embodiment the plant or part thereof is selected from the groupof monocotyledonous plants, dicotyledonous plants, annual, biannual andperennial plants, vegetable plants or harvested vegetables, fruit plantsor trees or harvested fruits, flower bearing plants or trees orharvested flowers, cereal plants, oleaginous plants, proteinous plants,ligneous plants, and ornamental plants.

In one embodiment the plant or part thereof is an agriculturallyimportant plant, cultivar thereof, or product thereof selected from thegroup consisting of agriculturally important vines, agriculturallyimportant vegetables and agriculturally important fruit plants, andcultivars and products thereof. Preferably the agriculturally importantvine is a kiwifruit vine or cultivar thereof, and the product iskiwifruit.

In one embodiment the kiwifruit vine is selected from the groupconsisting of species of green-fleshed kiwifruit (A. chinensis var.deliciosa), golden kiwifruit (A. chinensis var. chinensis), Chinese egggooseberry (A. coriacea), baby kiwifruit (A. arguta), Arctic kiwifruit(A. kolomikta), red kiwifruit (A. melanandra, A. chinensis var.chinensis), silver vine (A. polygama), and purple kiwifruit (A.purpurea) or a cultivar thereof. Preferably the kiwifruit are selectedfrom the group consisting of A. chinensis var. deliciosa and A.chinensis var. chinensis, species or a cultivar thereof. Preferably thekiwifruit is a species of A. chinensis var. chinensis. Preferably thekiwifruit is A. chinensis var. chinensis Planch. Preferably the cultivaris a ‘Hayward’ or ‘Hort16A’ or ‘zesy002’, informally known as Gold3 or‘Hongyang’.

In one embodiment the cultivar is A. chinensis var. chinensis Planch,‘Hort16A’. In one embodiment the cultivar is ‘Hort16A’ as disclosed inUSPP11066, the entirety of which is incorporated by reference herein.

In one embodiment the cultivar is A. chinensis var. deliciosa ‘Hayward’.In one embodiment the cultivar is ‘Hayward’ as disclosed in USPP6815,the entirety of which is incorporated by reference herein.

In one embodiment cultivar is A. chinensis var. chinensis Planch.‘Hongyang’. In one embodiment the cultivar is ‘Hongyang’ as disclosed inWang 2011 and in Li et al 2015, the entirety of which are incorporatedby reference herein.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for controlling Pseudomonasspp. bacteria on a plant or part thereof.

The use of YBCA5, or a composition comprising YBCA5 for controllingPseudomonas spp., bacteria and/or for increasing the yield of akiwifruit plant is carried out in accordance with the methods of theinvention as described herein. For example, YBCA5 and compositionsthereof may be prepared, formulated for and applied to a plant, or partthereof, particularly a kiwifruit plant, or part thereof, according tothe invention as described herein.

In another aspect the invention relates to a method of controlling atleast one phytopathogenic fungus on a plant or part thereof, the methodcomprising contacting the plant or part thereof with YBCA5, or acomposition comprising YBCA5.

In another aspect the invention relates to a method for increasing theyield of a fruit or vegetable plant susceptible to infection by aphytopathogenic fungus, the method comprising applying YBCA5, or acomposition comprising YBCA5 to the fruit or vegetable plant or partthereof, and growing the plant or part thereof. In one embodiment thecomposition consists essentially of YBCA5.

In one embodiment the at least phytopathogenic fungus is selected fromthe group consisting of Botrytis spp., Monilinia spp., Sclerotinia spp.,Colletotrichum spp., Alternaria spp., Cryptosporiopsis spp., Phomopsisspp., and Penicillium spp.

In one embodiment the plant or part thereof is selected from the groupof monocotyledonous plants, dicotyledonous plants, annual, biannual andperennial plants, vegetable plants or harvested vegetables, fruit plantsor trees or harvested fruits, flower bearing plants or trees orharvested flowers, cereal plants, oleaginous plants, proteinous plants,ligneous plants, and ornamental plants.

In one embodiment the plant or part thereof is an agriculturallyimportant plant, cultivar thereof, or product thereof selected from thegroup consisting of agriculturally important vines and agriculturallyimportant fruit trees, agriculturally important vegetables and cultivarsand products thereof. In one embodiment the agriculturally importantvine is a kiwifruit vine or cultivar thereof, and the product iskiwifruit.

In one embodiment the plant or part thereof is a fruit or vegetableplant or part thereof, the method comprising contacting the fruit orvegetable plant or part thereof with YBCA5, or a composition comprisingYBCA5. In some embodiments the fruit or vegetable plant is a cherry treeor a grape vine. In some embodiments the fruit plant is an apple tree.

In one embodiment the cherry tree is a Prunus spp., or a cultivarthereof, preferably a P. avium, or cultivar thereof. Preferably the P.avium is a “Sweet Valentine” variety. In one embodiment the part thereofis a flower or part thereof or a fruit or part thereof. In oneembodiment the fruit is a cherry.

In one embodiment the grape vine is a Vinus spp., or a cultivar thereof,preferably a V. vinifera, or cultivar thereof. Preferably the V.vinifera is a “Thompson Seedless” variety. In one embodiment the partthereof is a flower or part thereof or a fruit or part thereof. In oneembodiment the fruit is a grape.

In one embodiment the apple tree is a Malus spp., or a cultivar thereof,preferably M. pumila or cultivar thereof. Preferably the M. pumila or acultivar thereof is a ‘Pacific Rose’ variety. In one embodiment the partthereof is a flower or part thereof, or a fruit or part thereof. In oneembodiment the fruit is an apple.

Psa Control

In another aspect the invention relates to a method for controlling P.syringae pv. actinidiae (Psa) on a kiwifruit plant or part thereof, themethod comprising contacting the kiwifruit plant or part thereof withYBCA5, or a composition comprising YBCA5.

In another aspect the invention relates to a method for increasing theyield of a kiwifruit plant infected, or susceptible to infection withPsa, the method comprising applying YBCA5 or a composition comprisingYBCA5 to the kiwifruit plant or part thereof, and growing the kiwifruitplant or part thereof.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for controlling Psa on a kiwifruit plant orpart thereof.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for increasing the yield of a kiwifruitplant infected, or susceptible to infection with Psa.

In one embodiment the composition consists essentially of YBCA5.

In one embodiment, the kiwifruit plant is a species of A. chinensis var.deliciosa or A. chinensis var. chinensis, or a cultivar thereof,preferably a species of A. chinensis var. chinensis, or cultivarthereof. In one embodiment the kiwifruit plant is ‘Hort16A’.

In one embodiment cultivar is A. chinensis var. chinensis ‘Hongyang’. Inone embodiment the cultivar is ‘Hongyang’ as disclosed in Wang 2011 andin Li et al 2015, the entirety of which are incorporated by referenceherein.

In one embodiment a plant or part thereof is contacted for a timesufficient to control Psa.

In one embodiment, contacting comprises applying YBCA5 or a compositioncomprising or consisting essentially of YBCA5 to the plant or partthereof by applying to the seeds, leaves, stems, flowers, fruits, trunksand/or roots of the plant or part thereof. Preferably application is byspraying, misting, dipping, dripping, dusting or sprinkling.Applications can be made once only, or repeatedly as required. Alsocontemplated herein is application at various times of year and/orduring various stages of the plant life cycle, as determined appropriateby the skilled worker.

YBCA5 may be applied at the appropriate time during the year and at theappropriate stage of plant development as may be determined by a skilledworker. For example YBCA5 may be applied from bud-burst to flowering,during flowering and post flowering/fruit set period but not limitedthereto.

In one embodiment, applying is by spraying onto leaf surfaces and/oronto flowers and/or onto fruit and/or onto vegetables.

In one embodiment, applying to the roots is by ground spraying,mechanical incorporation or by mixing with enriching agents orfertilizers prior to application in the usual way.

In one embodiment the plant or part thereof is selected frommonocotyledonous plants, dicotyledonous plants, annual, biannual andperennial plants, vegetable plants or harvested vegetables, fruit plantsor trees or harvested fruits, flower bearing plants or trees orharvested flowers, cereal plants, oleaginous plants, proteinous plants,ligneous plants, and ornamental plants.

In one embodiment, a plant or part thereof is an agriculturallyimportant crop plant, cultivar or product thereof selected from cornplants, tobacco plants, wheat plants, sugar cane plants, rapeseedplants, barley plants, rice plants, sorghum plants, millet plants, soyabean plants, lettuce plants, and cabbage plants.

In one embodiment the plant or part thereof is an agriculturallyimportant plant, cultivar thereof, or product thereof selected from thegroup consisting of agriculturally important vines and agriculturallyimportant fruit trees, and cultivars and products thereof.

Preferably the agriculturally important fruit trees or cultivars thereofare selected from olive trees, apple trees, pear trees, citrus fruittrees, banana trees, pineapple trees, peach trees, apricot trees, cherrytrees, walnut trees and hazelnut trees and the products thereof areolives, apples, pears, citrus fruits, bananas, pineapples, peaches,apricots, cherries, walnuts and hazelnuts respectively. Preferably theagriculturally important vines or cultivars thereof are selected frompotato vines, beetroot vines, bean vines, pea vines, tomato vines,cucumber vines, melon vines, berry vines, grape vines and kiwifruitvines and the products thereof are potatoes, beetroots, beans, peas,tomatoes, cucumbers, melons, berries, grapes and kiwifruitsrespectively. Preferably the agriculturally important vine is akiwifruit vine or cultivar thereof, and the product is kiwifruit.

Kiwifruit are within the plant order Ericales and the familyActinidiaceae. In one embodiment the kiwifruit vine is selected from thegroup consisting of species of fuzzy kiwifruit (A. chinensis var.deliciosa), golden kiwifruit (A. chinensis var. chinensis), Chinese egggooseberry (A. coriacea), baby kiwifruit (A. arguta), Arctic kiwifruit(A. kolomikta), red kiwifruit (A. melanandra, A. chinensis var.chinensis), silver vine (A. polygama), and purple kiwifruit (A.purpurea) or a cultivar thereof. Preferably the kiwifruit are selectedfrom the group consisting of A. chinensis var. deliciosa, A. chinensisvar. chinensis species or a cultivar thereof. Preferably the kiwifruitis a species of A. chinensis var. chinensis. Preferably the preferablykiwifruit is A. chinensis var. chinensis Planch. Preferably the cultivaris a ‘Hayward’ or ‘Hort 16A’ or ‘Zesy002’ or ‘Zesy004’ or ‘Hongyang’variety cultivar.

In one embodiment the cultivar is A. chinensis var. chinensis Planch,‘Hort 16A’. In one embodiment the cultivar is ‘Hort 16A’ as disclosed inUSPP11066, the entirety of which is incorporated by reference herein.

In one embodiment cultivar is A. chinensis var. deliciosa ‘Hayward’. Inone embodiment the cultivar is ‘Hayward’ as disclosed in USPP6815, theentirety of which is incorporated by reference herein.

In one embodiment cultivar is A. chinensis var. chinensis ‘Hongyang’. Inone embodiment the cultivar is ‘Hongyang’ as disclosed in Wang 2011 andin Li et al 2015, the entirety of which are incorporated by referenceherein.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for controlling Psa on akiwifruit plant or part thereof.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for increasing the yield of akiwifruit plant infected, or susceptible to infection with Psa.

The use of YBCA5, or a composition comprising YBCA5 for controlling Psaand/or for increasing the yield of a kiwifruit plant is carried out inaccordance with the methods of the invention as described herein. Forexample, YBCA5 and compositions thereof may be prepared, formulated forand applied to a plant, or part thereof, particularly a kiwifruit plant,or part thereof, according to the invention as described herein.

In another aspect the invention relates to at least one plant or partthereof treated with YBCA5, or a composition comprising YBCA5. In someembodiments the plant is a fruit or vegetable plant or part thereof. Inone embodiment the plant is a kiwifruit vine, a cherry tree or a grapevine.

Phytopathogenic Fungal Control

In another aspect the invention relates to a method of controlling atleast one phytopathogenic fungus on a plant or part thereof, the methodcomprising contacting the plant or part thereof with YBCA5, or acomposition comprising YBCA5.

In another aspect the invention relates to a method for increasing theyield of a fruit or vegetable plant susceptible to infection by aphytopathogenic fungus, the method comprising applying YBCA5, or acomposition comprising YBCA5 to the fruit or vegetable plant or partthereof, and growing the plant or part thereof.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for controlling a phytopathogenic fungus ona fruit or vegetable plant or part thereof.

In another aspect the invention relates to the use of YBCA5, or acomposition comprising YBCA5 for increasing the yield of a fruit orvegetable plant or part thereof susceptible to infection by aphytopathogenic fungus.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for controlling at least onephytopathogenic fungus on a plant or part thereof.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for controlling at least onephytopathogenic fungus on a fruit or vegetable plant or part thereof.

In another aspect the invention relates to YBCA5, or a compositioncomprising YBCA5 for use in, or when used, for increasing the yield of afruit or vegetable plant susceptible to infection by at least onephytopathogenic fungus.

The following embodiments are also specifically contemplated for thoseaspects of the invention that relate to the use of YBCA5, or to acomposition comprising or consisting essentially of YBCA5, forcontrolling phytopathogenic fungi and/or for increasing the yield of aplant or part thereof, or of a fruit or vegetable plant or part thereof,susceptible to infection by a phytopathogenic fungus.

In one embodiment the phytopathogenic fungus is selected from the groupconsisting of Botrytis spp., Monilinia spp., Sclerotinia spp.,Colletotrichum spp., Alternaria spp., Cryptosporiopsis spp., Phomopsisspp., and Penicillium spp.

In one embodiment the plant or part thereof is selected from the groupof monocotyledonous plants, dicotyledonous plants, annual, biannual andperennial plants, vegetable plants or harvested vegetables, fruit plantsor trees or harvested fruits, flower bearing plants or trees orharvested flowers, cereal plants, oleaginous plants, proteinous plants,ligneous plants, and ornamental plants.

In one embodiment the plant or part thereof is an agriculturallyimportant plant, cultivar thereof, or product thereof selected from thegroup consisting of agriculturally important vines and agriculturallyimportant fruit trees, agriculturally important vegetables and cultivarsand products thereof. In one embodiment the agriculturally importantvine is a kiwifruit vine or cultivar thereof, and the product iskiwifruit.

In one embodiment the plant or part thereof is a fruit or vegetableplant or part thereof, the method comprising contacting the fruit orvegetable plant or part thereof with YBCA5, or a composition comprisingYBCA5. In some embodiments the fruit or vegetable plant is a cherry treeor a grape vine. In some embodiments the fruit plant is an apple tree.

In one embodiment the cherry tree is a Prunus spp., or a cultivarthereof, preferably a P. avium, or cultivar thereof. Preferably the P.avium is a “Sweet Valentine” variety. In one embodiment the part thereofis a flower or part thereof or a fruit or part thereof. In oneembodiment the fruit is a cherry.

In one embodiment the grape vine is a Vinus spp., or a cultivar thereof,preferably a V. vinifera, or cultivar thereof. Preferably the V.vinifera is a “Thompson Seedless” variety. In one embodiment the partthereof is a flower or part thereof or a fruit or part thereof. In oneembodiment the fruit is a grape.

In one embodiment the apple tree is a Malus spp., or a cultivar thereof,preferably M. pumila or a cultivar thereof. Preferably the M. pumila isa ‘Pacific Rose’ variety. In one embodiment the part thereof is a floweror part thereof, or a fruit or part thereof. In one embodiment the fruitis an apple.

In another aspect the invention relates to at least one plant or partthereof treated with YBCA5, or a composition comprising YBCA5.

In another aspect the invention relates to at least one fruit orvegetable plant or part thereof treated with YBCA5, or a compositioncomprising YBCA5.

In one embodiment the composition consists essentially of YBCA5.

The use of YBCA5, or a composition comprising or consisting essentiallyof YBCA5 for controlling phytopathogenic fungi and/or for increasing theyield of a plant or part thereof, or of a fruit or vegetable plant orpart thereof is carried out in accordance with the methods and uses ofthe invention as described herein. For example, YBCA5 and compositionsthereof may be prepared, formulated for and applied to a plant, or partthereof, particularly a fruit or vegetable plant, or part thereof,particularly a cherry tree or grape vine, according to the invention asdescribed herein.

Various aspects of the invention will now be illustrated in non-limitingways by reference to the following examples.

EXAMPLES Example 1—Identification of Yeasts with Biocontrol Activity

Yeast Screening

YBCA5 was isolated from Apricots (“Clutha Gold”) from central Otago inthe early 2000s as follows. Fresh, harvested apricots were frozenovernight at −20° C. and then incubated at 20° C. for up to 5 days.Yeast or yeast-like colonies growing on the surface of selected apricotswere isolated using standard protocols on a general culturing mediumsuitable for yeast propagation.

Example 2—Yeast Biocontrol of Pseudomonas syringae var. actinidiae (Psa)

General Methodologies

Plant-based screening assays were conducted in laboratories andglasshouses at the Ruakura Research Centre, Hamilton and at the Te PukeResearch Orchard, Te Puke, New Zealand. Plant and Food (PFR) assaysfocused on foliar application of biological control agents (BCAs),particularly YBCA5 and other PFR proprietary yeast strains.

Zespri Assay 26—Dose Rate of YBCA5

The aim of this assay was to compare several dose rates of freshlyfermented YBCA5 with a formulated and dried preparation of YBCA5 fortheir efficacy against Psa.

Plant Material

Zespri Assay 26 was carried out in the PC1 glasshouse at Ruakura usingtissue cultured A. chinensis var. deliciosa ‘Hayward’ plants grown in 1L pots. Plants were 30-50 cm high, each with at least 4-5 useable leavesper plant and the time of treatment and there were 10 replicate plantsper treatment.

Yeast Preparation

Freshly fermented YBCA5 was obtained by fermenting the yeast for 3 daysin a 10 L bioreactor (Labfors) using sterile liquid media (4% molassesand 1.2 g/L urea). The fermentate was spun in a centrifuge (SorvallRC-5C) at 5000 rpm for 15 min (rotor no. SLC-4000, rotorcode 33) toachieve a wet pellet of cell concentrate after discarding thesupernatant. A sub-sample of wet pellet was re-suspended and the celldensity determined with the aid of a haemocytometer and appropriatedilutions made to achieve final spray concentrations of 6×10⁶, 1.25×10⁷,2.5×10⁷ and 5.0×10⁷ CFU/mL.

YBCA5 granules were prepared by mixing the wet pellet from a previousfermentation in the 10 L fermenter, with approximately 30% (w/w)cornstarch to form a stiff dough consistency and this was extrudedthrough a steel mesh (3 mm hole size) and dried in a laminar flow hoodovernight (20-25° C.) to form dried granules.

The number of CFU in the dried granules of YBCA5 was calculated bythoroughly dissolving 0.2 g granule into 20 mL of PBSTw. Serialdilutions of this stock were carried out (to 10-6) and three 10 μldroplets of each dilution were transferred onto MYA. The number of yeastcolonies growing from each droplet were counted after 24 h incubation at25° C. followed by a further 24 h incubation in a fridge (4-6° C.). Aspray concentration of 2.5×10⁷ CFU/mL was prepared be weighing theappropriate quantity of granules into 500 mL water.

Psa Inoculum Preparation

A Psa culture (isolate code 10627, biovar 3), which had been isolatedfrom an infected Actinidia chinensis var. chinensis ‘Hort16A’ kiwifruitvine located in the Te Puke region during 2010, was used for all staband spray inoculation assays included in this report (Vanneste et al.2013). Psa inoculum was prepared by growing this strain of Psa for 2-3days on King's B (KB) medium and harvesting the bacteria by washing theplate with sterile distilled water (SDW) to make a stock suspension ofinoculum that was visually determined to be c. >1×10⁹ CFU/mL. Asubsample of this Psa stock was serially diluted and 10-4 dropletsplaced onto fresh KB medium so that the number of CFU/mL could becounted after two days' incubation. To facilitate droplet inoculation inthe glasshouse (Ruakura), Psa inoculum (stock solution c.×10⁹ CFU/mL)optical density was determined using a spectrophotometer (600 nm) andthen the solution was diluted with sterile PBS to give resultantsuspensions of 5×10⁸ CFU/mL and 2×10⁷ CFU/mL, based on a previouslydeveloped calibration curve. The adjuvant, Du-Wett was then added to thesuspension to give a final concentration of 0.03% (v/v).

Leaf Spray Inoculation Assay

On 11 and 18 Sep. 2014, spray treatments of each freshly fermented YBCA5concentration and the granule preparation were applied to run-off to allleaves on each plant that had been grown in pots in a glasshouse, 7 and1 days before inoculation (dbi) with the two doses of Psa and plantswere allowed to dry in a spray containment shed. Once dried, the spraytreated plants were returned to the glasshouse. One day after the secondspray treatment with YBCA5, on 19 Sep. 2014 the Psa inoculum waspipetted (10 μL) onto the underside of each leaf in pairs on either sideof the mid-rib of four or five selected leaves (avoiding the oldest andthe youngest leaves). Plants were then placed into high-humidity tentsin containment glasshouses at PFR Ruakura after inoculation with Psa forup to three weeks and then scored for Psa severity.

Measurement of Psa Symptoms

The area (mm²) of necrosis caused by Psa was visually estimated for eachinoculation point 21 days after Psa droplet inoculation. In order toensure consistency, only two staff members carried out Psa leaf severityassessments with regular cross-checking of the severity scores.

Statistical Analysis

All data were analysed using GenStat following natural logtransformation. Raw data means are presented and statistical differencesare based on the log transformed analysis.

KRIP-BCA Assay 39

The aim of this assay was to investigate the fermentation, formulationand efficacy against Psa of different isolates of Aureobasidiumpullulans compared with YBCA5.

Plant Material

KRIP-BCA Assay 39 was carried out in the PC1 glasshouse at Ruakura usingtissue cultured A. chinensis var. deliciosa ‘Hayward’ plants grown in1.5 L pots. Plants were 30-50 cm high, each with at least 4-5 useableleaves per plant and the time of treatment and there were 10 replicateplants per treatment.

Yeast Preparation

YBCA5 granules were prepared by fermenting the yeast for 3 days in a 10L bioreactor (Labfors) using sterile liquid media (4% molasses and 1.2g/L urea). The fermentate was spun in a centrifuge (Sorvall RC-5C) at5000 rpm for 15 min (rotor no. SLC-4000, rotorcode 33) to achieve a wetpellet of cell concentrate after discarding the supernatant. This wetpellet was mixed with approximately 30% (w/w) cornstarch to form a stiffdough consistency and this was extruded through a steel mesh (3 mm holesize) and dried in a laminar flow hood overnight (20-25° C.) to formdried granules.

Twelve A. pullulans isolates selected from a large culture collectionwere fermented for 3 days in flask culture (200 mL of 4% molasses 1.2g/L urea sterile liquid media in 500 mL conical flasks). The number ofviable colony forming units (CFU) was determined by sub-sampling 1 mL offermentate and carrying out serial dilutions (to 10-7) in 0.05Mphosphate buffered saline +0.05% Tween80 (PBSTw). For each dilutionthree 10 μl droplets were transferred onto Malt Yeast Extract Agar(MYA). This number of yeast colonies growing from each droplet werecounted after 24 h incubation at 25° C. followed by a further 24 hincubation in a fridge (4-6° C.). The fermentate was then processed asdescribed above for YBCA5 to form dried formulated granules.

The number of CFU in the dried granules for each A. pullulans isolatewas calculated by thoroughly dissolving 0.2 g granule into 20 mL ofPBSTw. Serial dilutions of this stock were carried out (to 10-6) andthree 10 μl droplets of each dilution were transferred onto MYA. Thisnumber of yeast colonies growing from each droplet were counted after 24h incubation at 25° C. followed by a further 24 h incubation in a fridge(4-6° C.). Compositions for spray applications were prepared by weighingthe appropriate quantity of granules into 500 mL water.

Psa Inoculum Preparation

A Psa culture (isolate code 10627, biovar 3), which had been isolatedfrom an infected Actinidia chinensis var. chinensis ‘Hort16A’ kiwifruitvine located in the Te Puke region during 2010, was used for all staband spray inoculation assays included in this report (Vanneste et al.2013). Psa inoculum was prepared by growing this strain of Psa for 2-3days on King's B (KB) medium and harvesting the bacteria by washing theplate with sterile distilled water (SDW) to make a stock suspension ofinoculum that was visually determined to be c. >1×10⁹ CFU/mL. Asubsample of this Psa stock was serially diluted and 10 μL dropletsplaced onto fresh KB medium so that the number of CFU/mL could becounted after two days' incubation. To facilitate droplet inoculation inthe glasshouse (Ruakura), Psa inoculum (stock solution c.×10⁹ CFU/mL)optical density was determined using a spectrophotometer (600 nm) andthen the solution was diluted with sterile PBS to give resultantsuspensions of 1×10⁸ CFU/mL, based on a previously developed calibrationcurve. The adjuvant, Du-Wett was then added to the suspension to give afinal concentration of 0.03% (v/v).

Leaf Spray Inoculation Assays

On 9 May 2016, spray treatments of each A. pullulans isolate and YBCA5were applied to run-off to all leaves on each plant that had been grownin pots in a glasshouse, 7 days before inoculation (dbi) with Psa (1×10⁸CFU/mL). All A. pullulans treatments, including YBCA5 were applied at afinal concentration of 2×10⁷ CFU/mL and plants were allowed to dry in aspray containment shed. Once dried, the spray treated plants werereturned to the glasshouse. Seven days after spray treatment, on 16 May2016 the Psa inoculum dose was pipetted (10 μL) onto the underside ofeach leaf in pairs on either side of the mid-rib of four or fiveselected leaves (avoiding the oldest and the youngest leaves). Plantswere then placed into high-humidity tents in containment glasshouses atPFR Ruakura after inoculation with Psa for up to three weeks and thenscored for Psa severity.

Measurement of Psa Symptoms

The area (mm²) of necrosis caused by Psa was visually estimated for eachinoculation point 21 days after Psa droplet inoculation. In order toensure consistency, only two staff members carried out Psa leaf severityassessments with regular cross-checking of the severity scores.

Statistical Analysis

All data were analysed using GenStat following natural logtransformation. Raw data means are presented and statistical differencesare based on the log transformed analysis.

Results—Dose Rate Assay

FIG. 1 shows that YBCA5 is very effective at reducing the severity ofPsa symptoms on ‘Hayward’ kiwifruit leaves. All dose rates used in thisexperiment significantly reduced (P<0.001) the severity of leaf necrosiscompared with the untreated control. There was not difference inefficacy in the YBCA5 granule preparation compared with freshlyfermented YBCA5.

Results—Fermentation and Formulation

The fermentation yield for the 12 flask grown A. pullulans isolatesranged from 1.3×10⁸ CFU/mL to 2.3×10⁹ CFU/mL and the fermentation yieldfor flask grown YBCA5 was 3.3 ×10⁸ CFU/mL (Table 1), indicating thatsome isolates are capable of producing higher fermentation yieldscompared with YBCA5, while others produce lower fermentation yields.

The number of CFU/g for dried granules of YBCA5 (from 10 L fermentation)was 2.3×10¹⁰ and for the 12 isolates of A. pullulans the number of CFU/gfor dried granules ranged from a low of 3.1×10⁹ CFU/g to 2.0×10¹⁰ CFU/g(Table 1), indicating that most A. pullulans isolates produced a loweryield of viable CFU/g than YBCA5.

A comparison of the number of CFU in the granules per mL of fermentationliquid (to allow a more direct comparison of the 10 L fermentation ofYBCA5 and the flask culture of the 12 A. pullulans isolates) shows thatthe YBCA5 has the highest yield (4×10⁸ CFU/mL) and for the other A.pullulans isolates this ranged from as low as 4.2×107 CFU/mL to 2.4×10⁸CFU/mL (Table 1).

TABLE 1 Fermentation yield and formulation yield for a range ofAureobasidium pullulans isolates, including YBCA5. A. Conical GranulesGranule pullulans flask dried CFU/mL of culture yield weight GranuleTotal fermentation code (spores/mL) (g) CFU/g spores liquid YBCA53.3E+08 174* 2.30E+10* 4.04E+12* 4.0E+08* CG 173 ND**  9 2.00E+101.80E+11 2.2E+08 HB 229 ND  9.4 1.40E+10 1.30E+11 1.6E+08 HRY 212 ND 7.6 1.80E+10 1.40E+11 1.7E+08 HB 228 ND  9.8 1.90E+10 1.90E+11 2.4E+08HB226 2.3E+09  12 1.60E+10 1.90E+11 2.4E+08 HB201 1.4E+08  9.9 6.10E+096.00E+10 7.5E+07 FOR 5-8-1 2.3E+09  10 1.60E+10 1.60E+11 2.0E+08 GIS 084/1 2.3E+09  13.7 5.30E+09 7.30E+10 9.1E+07 HB 303 8.8E+09  10.83.10E+09 3.40E+10 4.2E+07 FOR6-1-1 1.3E+08  10.7 9.80E+09 1.10E+111.4E+08 HBR018 2.3E+08  7.9 1.50E+10 1.20E+11 1.5E+08 MSB 8-6-2 2.6E+08 8 1.10E+10 8.80E+10 1.1E+08 *For YBCA5 this data is the mean of threebatches fermented using the 10 L fermenter. **ND = No data.

Results—Leaf Droplet Inoculation Assay—Efficacy (KRIP-BCA 39)

In the Nil (wetter only) treatment, the average Psa lesion area was 57mm² (FIG. 2). One isolate (MSB8-6-2) did not significantly reduce(P>0.05) Psa severity (lesion size=54 mm²) compared to the Nil control.YBCA5 significantly reduced Psa lesion area to 43 mm2(efficacy=25%).This assay demonstrated that not all Aureobasidiumpullulans isolates have the ability to significantly reduce Psa severityon potted kiwifruit plants and efficacy against Psa is dependent uponthe isolate selected.

Zespri Assay 31

The aim of this assay was to compare the efficacy of YBCA5 applied aloneand integrated with copper or Actigard for control of Psa on pottedplants exposed to natural Psa inoculum in a research orchard.

Plant Material

This assay was carried out in the shadehouse structure (Block 20) at TePuke Research Orchard. The plants were originally grown at the Ruakuraglasshouse using tissue cultured A. chinensis var. deliciosa ‘Hayward’plants grown in 1.5 L pots. Once the plants were 25 cm in height theywere re-potted onto 2.5 L pots and moved to the Ruakura shade house andconnected up to dripper irrigation on 30 Oct. 2015. At the time oftreatment on 3 Nov. 2015, each plant had at least 4 useable leaves perplant and there were 15 replicate plants per treatment. Treatments andfoliar spray dates are described in Table 2.

TABLE 2 Treatment schedule for potted ‘Hayward’ plants exposed tonatural Psa inoculum at the Te Puke Research Orchard. 2^(st) 3^(rd)4^(th) 1^(st) Foliar Foliar Foliar TRT Foliar Treatment TreatmentTreatment No. treatment (+10d) (+10d) (+10d) Site Ruakura TPRO TPRO TPROSpray Nov. 3, Nov. 13, Nov. 23, Dec. 3, date 2015 2015 2015 2015Rationale 1. Nu-Film* Nu-Film Nu-Film Nu-Film Wetter control Copper 2.Kocide** Kocide Kocide Kocide based std 3. YBCA5 YBCA5 YBCA5 YBCA5 YBCA54. Kocide YBCA5 Kocide YBCA5 Integrated programme I 5. Actigard YBCA5Actigard YBCA5 Integrated programme II *Miller Chemical & FertilizerCorporation, USA **DuPont, USA

Yeast Preparation

YBCA5 granules were prepared by fermenting the yeast for 3 days in a 10L bioreactor (Labfors) using sterile liquid media (4% molasses and 1.2g/L urea). The fermentate was spun in a centrifuge (Sorvall RC-5C) at5000 rpm for 15 min (rotor no. SLC-4000, rotorcode 33) to achieve a wetpellet of cell concentrate after discarding the supernatant. This wetpellet was mixed with approximately 30% (w/w) cornstarch to form a stiffdough consistency and this was extruded through a steel mesh (3 mm holesize) and dried in a laminar flow hood overnight (20-25° C.) to formdried granules.

All YBCA5 treatments were applied at a final concentration of 2×10⁷CFU/mL and plants were allowed to dry. The final volume that wasprepared ranged from 500 mL to one litre depending on the size of theplants being treated.

Psa Inoculum Preparation

The aim of this project was to expose potted plants to Psa inoculum inat the Te Puke

Research Orchard (Block 20). This block was surrounded by maturekiwifruit vines with a history of Psa and this provided the inoculumover the period of time for this assay.

Leaf Sprays

The first spray treatments were applied at Ruakura on 3 Nov. 2016 and at10-14 day intervals thereafter. (Details are described in Table 2above). All treatments were applied to just prior to run-off with a handheld pump sprayer. Copper hydroxide (Kocide Opti) as applied at 0.7 g/Land Actigard was applied at 0.1 g/L. YBCA5 was applied with thewetter/sticker adjuvant, Nu-Film (250 ul per 500 ml).

Disease assessments were carried out on 17 Dec. 2015 by estimating thepercentage area of leaf necrosis on all treated leaves.

Results—Assay Zespri 31

In the absence of any treatment (Nu-film only, control), Psa leaf spotincidence was 66% and this was significantly reduced by Kocide Opti(18%), YBCA5 (35%), YBCA5 and Kocide Opti (Integrated programme I)(15%), YBCA5 and Actigard (Integrated programme II) (22%) (FIG. 3).

This assay demonstrated that YBCA5 significantly reduced (P<0.05) theincidence of Psa leaf spotting on potted plants under a shaded structurewhen exposed to natural Psa inoculum. Although the level of diseasecontrol was not as effective as the Kocide Opti based programme, theassay demonstrated that YBCA5 could be successfully integrated with acopper based product and with Actigard with no significant loss ofefficacy compared with the copper only treatment.

Example 3—Yeast Biocontrol of Phytopathogenic Fungi

YBCA5 Biocontrol of Monilinia fructicola and Botrytis spp.

Methods

Fruit-based screening assays were conducted in laboratories at the Plantand Food Research Ruakura Research Centre, Hamilton, New Zealand (PFR).PFR assays focused on dip treatment application of YBCA5 and fungicidecontrols.

Fruit Material (Assays 1 to 4)

Fruit for Monilinia fructicola and Botrytis spp. inoculation assays werecarried out on sweet cherries (Prunus avium ‘Sweet Valentine’) that werepicked at the harvest mature stage and sourced from the PFR ClydeResearch Orchard in Central Otago on 8 Jan. 2016 for fruit based assays1 and 2. A second harvest was carried out on 13 Jan. 2016 for fruitbased assays 3 and 4.

Each cherry was then subjected to a double wash process. Wash oneconsisted of 10 minutes in tap water on a rotary shaker (110 rpm)followed by a five minute wash in SDW (Wash 2). All cherries were placedonto sterile black plastic grids in a sterile plastic meat tray with twosterile paper towels and were allowed to dry in a laminar flow hood.Each cherry was dipped in the treatments for 60 seconds and againallowed to dry, as described above. 40 ml deionised water was added tothe paper towels to ensure high relative humidity, and then enclosed ina plastic bag to incubate at 23° C. for 24 h (Assay 1) and 48 hours(Assay 2) to allow the YBCA5 treatment to become established on thefruit surface.

Assay 5 Fruit Material

Fruit assays were carried out on detached white table grape berries(‘Thompson seedless’—Assay 5), imported from California that weresourced from a local supermarket in Hamilton. Each berry was detachedfrom the bunch with 3-4 mm of pedicel remaining and then subjected to adouble wash process. Wash one consisted of 10 minutes in tap water on arotary shaker (110 rpm) followed by a five minute wash in SDW (Wash 2).All berries were placed onto sterile black plastic grids in a sterileplastic meat tray with two sterile paper towels and were allowed to dryin a laminar flow hood. Each berry was lightly wounded with the aid offine sandpaper, Grade P220, then dipped in the treatments for 60 secondsand again allowed to dry.

YBCA5 preparation (Assays 1 to 5)

YBCA5 granules were prepared by fermenting the yeast for 3 days in a 10L bioreactor (Labfors) using sterile liquid media (4% molasses and 1.2g/L urea). The fermentate was spun in a centrifuge (Sorvall RC-5C) at5000 rpm for 15 min (rotor no. SLC-4000, rotorcode 33) to achieve a wetpellet of cell concentrate after discarding the supernatant. This wetpellet was mixed with approximately 30% (w/w) cornstarch to form a stiffdough consistency and this was extruded through a steel mesh (3 mm holesize) and dried in a laminar flow hood overnight (20-25° C.) to formdried granules.

The YBCA5 treatments were prepared from these water dispersible granulesthat had been stored at 5-7° C. in a refrigerator and a suspensionprepared by adding 1 g per litre of deionised water (finalconcentration=2×10⁷ CFU/ml) and gently stirred to form a suspension. Toensure all cells were evenly dispersed and remained in suspension, awetting agent (Nu-Film) was added at 0.5 ml per litre.

Fungicides (Assays 1 to 5)

For assays 1 and 2, a liquid suspension containing 500 g/L) was preparedat the recommended field rate of 0.85 mL/L (an average of therecommended field rates of 0.75 mL/L for Monilinia in stonefruit and 1.0mL/L for Botrytis in berryfruit). No wetting agent was used.

For assays 3 and 4, a liquid suspension of captan was prepared fromCaptan Flo (Nufarm NZ) (containing captan at 600 g/L) at the recommendedfield rate for use in stone fruit of 160 mL/litre. No wetter was used.

Monilinia inoculum Preparation (Assays 1 and 3)

A Monilinia fructicola culture (isolate code MFGQ3), which had beenoriginally isolated from an infected peach tree located in the Hamiltonregion during 1998, was used for the spray inoculation assays includedin this section (Ruakura based assay). Monilinia inoculum was preparedby growing this strain of Monilinia for 7 days on PDA (Difco, FortRichard) medium and harvesting the conidia by washing the plate with SDWplus Tween 80 (0.05%) to make a stock suspension of inoculum. This stocksuspension was then filtered using a 70 μm cell strainer (to removemycelial fragments), the concentration determined using a haemocytometerand then adjusted, by dilution with SDW+Tw 80 (0.05%) to a finalconcentration of 1×10⁴ conidia/mL.

Botrytis spp. inoculum Preparation (Assay 2 and 4)

A Botrytis spp. culture (isolate code 09-2), which had been originallyisolated from an infected kiwifruit located in the Bay of Plenty regionduring the 2000s, was used for the spray inoculation assays included inthis section carried out on cherries. Botrytis spp. inoculum wasprepared by growing this strain of Botrytis spp. for 5-7 days on PDA(DIFCO, Fort Richard) medium and harvesting the conidia by washing theplate with SDW plus Tween 80 (0.05%) to make a stock suspension ofinoculum. This stock suspension was then filtered using a 70 μm cellstrainer (70 μm mesh) to remove mycelial fragments, the concentrationdetermined using a haemocytometer and then adjusted, by dilution withSDW+Tw 80 (0.01% v/v) to a final concentration of 1×10⁵ conidia/mL.

Botrytis inoculum Preparation (Assay 5)

Two Botrytis spp. cultures (isolate codes 189 and 547), which had beenoriginally isolated from infected tomatoes, Auckland region during 2010,were used for the droplet inoculation assays included in this assay. Oneisolate was sensitive to two commonly used fungicides (dicarboximide andcarbendazim) and the other isolate was resistance to each of these samefungicides.

Botrytis spp. inoculum was prepared by growing each isolate of Botrytisspp. for 5-7 days on PDA (DIFCO, Fort Richard) medium and harvesting theconidia by washing the plate with SDW plus Tween 80 (0.01%) to make astock suspension of inoculum. This stock suspension was then filteredusing a cell strainer (70 μm mesh, Falcon) to remove mycelial fragments,the concentration determined using a haemocytometer and adjusted to therequired concentration (2×10⁴ conidia/mL) equivalent to 200 conidia ineach 10 μL droplet. To ensure the conidial suspension remained on thewounded berry surface, paraffin ‘wax bunds’ were created around thewound surface by smearing a 1-2 mm thick layer of paraffin wax onto aglass slide lightly dabbing the base of a 1 mL pipette tip onto theparaffin wax layer and then transferring this ‘paraffin wax ring’ ontothe wound surface. This effectively created a 5-6 mm diameter ring ofparaffin wax (‘bund’) that retained the conidial suspension andprevented it from rolling off the rounded berry surface. Wounded andtreated berries were then inoculated with 10 μL droplets of Botrytisspp. conidial suspension.

After pathogen inoculation, all inoculated fruit samples in a tray wereenclosed in a plastic bag for 48 hours on the lab bench at 23° C.(Assays 1-4) or 21° C. (Assay 5). For all cherry and berry assays, twosterile paper towels were placed beneath the plastic grids, on which theberries lay, then moistened with 40 mL SDW and each tray was enclosed ina clean plastic bag and then sealed to maintain high relative humidityover the first 72 h. Thereafter, the bags were removed, folded over thetrays (to ensure each end of the tray was open for adequate air flow),to allow the relative humidity to decline over a 15-h period (5 pm inthe late afternoon to 8 am the next morning) after which, they wereresealed. This process of alternating the relative humidity within theincubation chambers was repeated over the duration of the experiment andis a method that has been shown to avoid excessive mycelial growth.After 5 days incubation, fruit with typical Monilinia spp. or Botrytisspp. symptoms were recorded and removed from each replicate tray. Rotswere recorded daily and the incidence (%) of cumulative Monilinia spp.or Botrytis spp. rots, after 13 days (Assay 1 and 2), 16 days (Assay 3and 4) and 9 days (Assay 5). In assay 5, the severity of Botrytis spp.infection was determined for each treatment by visually scoring theproportion of the berry surface covered in Botrytis spp. conidiophores.

YBCA5 Biocontrol of Colletotrichum spp. and Penicillium spp. in Apples

Apple Assays 6 and 7

Apple fruit (‘Pacific Rose’) were sourced from an organic orchard inHawkes Bay and were washed in running tap water in a 10 L bucket. Appleswere then dried in a biohazard hood for approximately 1.5 h, turningthem after 45 min and then wiped with a tissue soaked in ethanol andallowed to dry once more. Apples were then placed onto moistened papertowels lining the bottom of plastic clam shell containers, two applesper container. There were 10 replicate apples for each treatment inassays 6 and 7.

There were five treatments: a Nil control (0.05% Tween80), Fungicide(0.5 mL/L of Prolific (containing 500 g/L carbendazim)), YBCA5 appliedat 1×10⁷ CFU/mL 24 hours prior to the pathogen, YBCA5 applied at 1×10⁷CFU/mL 2 hours prior to the pathogen and pathogen only (Colletotrichumspp. for assay 6 and Penicillium spp. for assay 7). YBCA5 granules wereprepared by fermenting the yeast for 3 days in a 10 L bioreactor(Labfors) using sterile liquid media (4% molasses and 1.2 g/L urea). Thefermentate was spun in a centrifuge (Sorvall RC-5C) at 5000 rpm for 15min (rotor no. SLC-4000, rotorcode 33) to achieve a wet pellet of cellconcentrate after discarding the supernatant. This wet pellet was mixedwith approximately 30% (w/w) corn-starch to form a stiff doughconsistency and this was extruded through a steel mesh (3 mm hole size)and dried in a laminar flow hood overnight (20-25° C.) to form driedgranules.

The YBCA5 treatments were prepared from these water dispersible granulesthat had been stored at 5-7° C. in a refrigerator and a suspensionprepared by adding 0.5 g per litre of deionised water (finalconcentration=1×10⁷ CFU/mL) and gently stirred to form a suspension.

The pathogen spore suspensions were prepared from cultures ofColletotrichum spp. and Penicillium spp. grown on PDA. One third of theculture was removed from the PDA Petri dish and transferred into a 50 mLFalcon tube containing 30 mL of SDW (with 0.05% Tween80). This wasshaken vigorously for 1 min to dislodge spores into the suspension andthen passed through a 70 μ cell strainer to remove any mycelialfragments. The spore concentration was calculated using the aid of ahaemocytometer and dilutions made to achieve a final concentration of1×10⁵ spores/mL

On the day the apples were washed and prepared (day 1), a small wound (3mm diameter×2-3 mm deep) were made in the side of each apple andnumbered as treatment 3. A 10 μ aliquot of YBCA5 suspension was added tothe wound, enough to fill the wound. On the following day (day 2) fourmore wounds were made and numbered as treatments 1, 2, 4 and 5.Treatments 1, 2 and 4 were applied to the wounds by adding 10 μLaliquots of each solution to the respective wounds. Two hours later thepathogen was applied by adding 10 μL aliquots of Colletotrichum spp. toeach wound (treatments 2, 3, 4 and 5) for assay 6 and adding 10 μLaliquots of Penicillium spp. to each wound (treatments 2, 3, 4 and 5)for assay 7. Thirty mL of additional SDW was added to the paper towel ineach clam shell container to maintain relative humidity and thecontainers were incubated in a Sanyo incubator at 23° C. for 1-2 weeksto allow rots to develop.

The lesion diameter of rots were measured using digital callipers after8 days. Lesion size was corrected for the diameter of the wound bysubtracting 3 mm from each measurement and then ANOVA was carried outusing Genstat to test for treatment differences based on leastsignificant differences.

Experimental Design

The cherry assays (assays 1-4) consisted of 10 cherries per replicateand there were six replicates (assays 1 and 2) and eight replicates(assays 3 and 4) for each treatment in a randomised block layout.

The grape berry assay (assay 5) consisted of five berries per replicateand there were four replicates for each treatment in a randomised blocklayout.

Statistical Analysis

Data were analysed using GenStat, 13th edition, with a randomised blockexperimental design and analysis of variance. Average fruit infection (%incidence) were log-transformed to equalise the variance to better meetthe normality assumptions of the analysis. Raw data means and LeastSignificant Differences (LSDs) are presented, however all statisticalcomparisons are based on the log analysis.

Results of Cherry Inoculation Assays

Assay 1

FIG. 9 summarises the effect of YBCA5 against Monilinia fruit rot incherries. The incidence of Monilinia fruit rot in the Nil treatment was50% and although the YBCA5 (34%) and iprodione treatments (22%) had alower incidence of Monilinia, these were not significant reductionscompared with the Nil treatment (FIG. 9).

Assay 2

FIG. 10 summarises the effect of YBCA5 against Botrytis spp. fruit rotin cherries. The incidence of Botrytis spp. fruit rot in the niltreatment was 35% and this was not significantly (P>0.05) reduced in theYBCA5 (24%) and iprodione treatment (22%) (FIG. 10).

Assay 3

FIG. 11 summarises the effect of YBCA5 against Monilinia fruit rot incherries in another assay (Assay 3). The incidence of Monilinia fruitrot in the nil treatment was 88% and this was not significantly (P>0.05)reduced by the YBCA5 (59%). The captan fungicide treatment (12%)significantly reduced (P<0.001) the incidence of Monilinia fruit rotcompared with the Nil treatment (FIG. 11).

Assay 4

FIG. 12 summarises the effect of YBCA5 against Botrytis spp. fruit rotin cherries in another assay. The incidence of Botrytis spp. fruit rotin the nil treatment was 67% and this was not significantly reduced(P>0.05) in the YBCA5 (49%) and captan treatment (43%) (FIG. 12).

Results of Grape Inoculation Assay

Assay 5

FIG. 13 summarises the effect of YBCA5 against Botrytis spp. fruit rotin table grapes in another assay (Assay 5). The incidence of Botrytisspp. fruit rot in the nil treatment 30% and this was significantly(P<0.001) reduced by the YBCA5 (7%) and captan treatment (10%) (FIG.13).

Treatment of Other Diseases—Apple Results for Apple Assays 6 and 7

Assay 6

The mean lesion size in the untreated Colletotrichum control was 10.1 mmand this was significantly (P<0.05) reduced in the fungicide and both ofthe YBCA5 treatments (Table 3).

Applying YBCA5 24 hrs before the pathogen provided better protectionthan applying 2 hrs before the pathogen.

TABLE 3 Average lesion size on wounded apples (“Pacific Rose”) treatedwith fungicide and YBCA5 prior to inoculation with Colletotrichum spp.spores (1 × 10⁵ spores/mL) and assessed after 8 days incubation at 23°C. Average lesion Treatment (day 8) Significance Nil (No Colletotrichum)0.3 d Fungicide (Prolific) 1.1 cd YBCA5 (24 hr) 2.2 c YBCA5 (2 hr) 5.7 bNil + Colletotrichum 10.1 a LSD (5%) 1.42 P value <0.001

Treatment means followed by different letters show significantdifference.

Assay 7:

The mean lesion size in the untreated Penicillium control was 15.2 mmand this was significantly (P<0.05) reduced in the fungicide and both ofthe YBCA5 treatments (Table 4). Similar to the previous assay, applyingYBCA5 24 h before the pathogen provided significantly better protectionthan applying YBCA5 2 h before the pathogen.

TABLE 4 Average Lesion size on wounded apples (“Pacific Rose” treatedwith fungicide and YBCA5 prior to inoculation with Penicillium spp.spores (1 × 10⁵ spores/mL) and assessed after 8 days incubation at 23°C. Treatment Average lesion Significance Nil (no Penicillium) 0.3 cFungicide (Prolific) 0.4 c YBCA5 24 h 0.4 c YBCA5 2 h 3.8 b Nil +Penicillium 15.2 a LSD (5%) 3.51 P value <0.001

Treatment means followed by different letters show significantdifference.

Assay 8: Post-Harvest Rot Due to Phytopathogenic Fungi on ‘Hongyang’Kiwifruit

The most important export cultivar from China is ‘Hongyang’ and this redand yellow-fleshed cultivar is attacked by a range of postharvestpathogens including Penicillium spp. Phomopsis spp., Alternaria spp.,Colletotrichum spp., Cryptosporiopsis spp. and Botrytis spp.

We investigated the efficacy of YBCA5 applied as a wound protectantagainst a range of postharvest fungal pathogens of fruits, particularly:Penicillium spp. Phomopsis spp., Alternaria spp., Colletotrichum spp.,Cryptosporiopsis spp. and Botrytis spp.

Preliminary tests were carried out to establish the concentration ofeach pathogen that was required to rot the fruit after wounding in theabsence of any treatment.

Methods

The kiwifruit ‘Hongyang’-based screening assays (assays 8 and 9) wereconducted in laboratories at the Plant and Food Research RuakuraResearch Centre, Hamilton, New Zealand (PFR). PFR assays focused onwound application of YBCA5 treatment, a commercial biological controltreatment and a fungicide were used as comparative controls.

Fruit Material (Assays 8)

The ‘Hongyang’ kiwifruit were sourced from the PFR Riwaka ResearchOrchard in

Motueka on 12 Apr. 2017 for fruit based assays 8 and 9. Penicillium spp.Phomopsis spp., Alternaria spp., Colletotrichum spp., Cryptosporiopsisspp. and Botrytis spp. inoculation assays were carried out on ‘Hongyang’kiwifruit that were picked at the harvest mature stage.

After removal from cool storage at 1° C., each fruit was subjected to atriple wash process. Wash one consisted of 30 seconds in 70% ethanolthen a wash in tap water for 10 minutes on a rotary shaker (80 rpm-Wash2) followed by a final wash for five minutes in SDW-Wash 3). All fruitwere placed onto sterile black plastic grids in a sterile plastic meattray with two sterile paper towels and were allowed to dry overnight ina laminar flow hood.

Just prior to wound treatment, each fruit was wounded on the side with asterile stainless steel spike (4 mm deep×3 mm wide) and 10 μI of eachtreatment suspension was pipetted into the wound and allowed to dry.

Treatments and Rates are Detailed Below

Treatment Recommended rate gm or mL/Litre YBCA5* 1 × 10⁷ 0.5 g/LSerenade Opti* 125 g/100 L 1.25 g/L Rovral Aquaflo 75 mL/100 L 0.75 mL/L*YBCA5 and Serenade Opti prepared in Tween 80 (0.05%), Rovral Aquaflowas prepared in deionized water.

For assays 8 and 9, two sterile paper towels were placed beneath Plixcut-outs, moistened with 40 mL SDW and each fruit placed in disposablelunch boxes (Plix Extra Deep 45/45, containing five Plix fruit cut-outsto prevent fruit from moving), sealed, then placed into large (40 L)plastic bins which were closed to ensure high relative humidity for thefirst 24 h and incubated on the lab bench at 24° C. After 24 h, the Plixlunch boxes were removed from the bins and a 10 ul suspension of eachfungal pathogen was pipetted into the treated wounds. All lunch boxeswere resealed and placed back into the large plastic bins to ensure highrelative humidity for the next 48 h. After this time, the Plix lunchboxes were removed from the large plastic bins, and a pin placed betweenthe Plix box lid and base to allow some air to circulate and therelative humidity to decline over a 15 h period (5 pm in the lateafternoon to 8 am the next morning) after which, they were resealedagain. This process of alternating the relative humidity within theincubation chambers was repeated over the duration of the experiment andis a method that has been shown to avoid excessive mycelial growth.After 6 days, (Alternaria spp. Botrytis spp., Penicillium spp.,Phomopsis spp., Colletotrichum spp.) and 7 days (Cryptosporiopsis spp.),the severity of fungal rot was assessed for each treatment by measuringthe lesion length (mm) along the axis of the fruit. Data were expressedas the average lesion length, minus the initial width of the wound (3mm).

YBCA5 Preparation (Assay 8 and 9)

YBCA5 granules were prepared by fermenting the yeast for 3 days in a 10L bioreactor (Labfors) using sterile liquid media (4% molasses and 1.2g/L urea). The fermentate was spun in a centrifuge (Sorvall RC-5C) at5000 rpm for 15 min (rotor no. SLC-4000, rotorcode 33) to achieve a wetpellet of cell concentrate after discarding the supernatant. This wetpellet was mixed with approximately 30% (w/w) corn-starch to form astiff dough consistency and this was extruded through a steel mesh (3 mmhole size) and dried in a laminar flow hood overnight (20-25° C.) toform dried granules.

The YBCA5 treatments were prepared from these water dispersible granulesthat had been stored at 5-7° C. in a refrigerator and a suspensionprepared by adding 0.5 g per litre of deionised water (finalconcentration=1×10⁷ CFU/ml) and gently stirred to form a suspension.

Postharvest Pathogen Preparation

Alternaria spp. inoculum Preparation (Assay 8)

The Alternaria spp. culture (isolate code=‘Alternaria ex cherry’), whichhad been originally isolated from an infected cherry fruit from CentralOtago during 2016, was used for the wound inoculation part of thisassay. Alternaria spp. inoculum was prepared by growing this strain ofAlternaria spp. for 21 days on Oat Meal Agar) medium and harvesting theconidia by washing the plate with SDW plus Tween 80 (0.05%) to make astock suspension of inoculum. This stock suspension was then filteredusing a 70 μm cell strainer (to remove mycelial fragments), theconcentration determined using a haemocytometer and then adjusted, bydilution with SDW+Tw 80 (0.05%) to a final concentration of 2×10⁴conidia/mL.

Botrytis spp. inoculum Preparation (Assay 8)

A Botrytis spp. culture (isolate code 09-2), which had been originallyisolated from an infected kiwifruit located in the Bay of Plenty regionduring the 2000s, was used for the spray inoculation assays included inthis section carried out on cherries. Botrytis spp. inoculum wasprepared by growing this strain of Botrytis spp. for 12 days on Oat MealAgar medium and harvesting the conidia by washing the plate with SDWplus Tween 80 (0.05%) to make a stock suspension of inoculum. This stocksuspension was then filtered using a 70 μm cell strainer (70 μm mesh) toremove mycelial fragments, the concentration determined using ahaemocytometer and then adjusted, by dilution with SDW+Tw 80 (0.01% v/v)to a final concentration of 1×10⁵ conidia/mL.

Colletotrichum spp. inoculum Preparation (Assay 8)

The Colletotrichum spp. culture (isolate code=‘ex G3), which had beenoriginally isolated from an infected Gold3 kiwifruit from the Ruakuraresearch orchard during 2017, was used for the wound inoculation part ofthis assay. Colletotrichum spp. inoculum was prepared by growing thisstrain of Colletotrichum spp. for 21 days on PDA (Difco, Fort Richard)medium and harvesting the conidia by washing the plate with SDW plusTween 80 (0.05%) to make a stock suspension of inoculum. This stocksuspension was then filtered using a 70 μm cell strainer (to removemycelial fragments), the concentration determined using a haemocytometerand then adjusted, by dilution with SDW+Tw 80 (0.05%) to a finalconcentration of 2×10⁴ conidia/mL.

Penicillium spp. inoculum Preparation (Assay 8)

The Penicillium spp. culture (isolate code=‘Penicillium ex lemon’),which had been originally isolated from an infected lemon fruit from asupermarket in 2017, was used for the wound inoculation part of thisassay. Penicillium spp. inoculum was prepared by growing this strain ofPenicillium spp. for 12 days on PDA (Difco, Fort Richard) medium andharvesting the conidia by washing the plate with SDW plus Tween 80(0.05%) to make a stock suspension of inoculum. This stock suspensionwas then filtered using a 70 μm cell strainer (to remove mycelialfragments), the concentration determined using a haemocytometer and thenadjusted, by dilution with SDW+Tw 80 (0.05%) to a final concentration of2×10⁴ conidia/mL.

Phomopsis spp. inoculum Preparation (Assay 8)

The Phomopsis spp. culture (isolate code=‘Phomopsis ex G3’), which hadbeen originally isolated from an infected Gold3 kiwifruit from theRuakura research orchard during 2017, was used for the wound inoculationpart of this assay. Phomopsis spp. inoculum was prepared by growing thisstrain of Phomopsis spp. for 21 days on PDA (Difco, Fort Richard) mediumand harvesting the conidia by washing the plate with SDW plus Tween 80(0.05%) to make a stock suspension of inoculum. This stock suspensionwas then filtered using a 70 μm cell strainer (to remove mycelialfragments), the concentration determined using a haemocytometer and thenadjusted, by dilution with SDW+Tw 80 (0.05%) to a final concentration of2×10⁴ conidia/mL.

Cryptosporiopsis spp. inoculum Preparation (Assay 9 Only)

The Cryptosporiopsis spp. culture (isolate code=‘Cryptosporiopsis exG3’), which had been originally isolated from an infected Gold3kiwifruit from the Te Puke Research Orchard was used for the woundinoculation in assay 9. Cryptosporiopsis spp. inoculum was prepared bygrowing this strain of Cryptosporiopsis spp. for 28 days on PDA (Difco,Fort Richard) medium and harvesting the conidia by washing the platewith SDW plus Tween 80 (0.05%) to make a stock suspension of inoculum.This stock suspension was then filtered using a 70 μm cell strainer (toremove mycelial fragments), the concentration determined using ahaemocytometer and then adjusted, by dilution with SDW+Tw 80 (0.05%) toa final concentration of 2×10⁴ conidia/mL.

After pathogen inoculation in assay 8 and 9, all inoculated fruitsamples were placed in disposable lunch boxes (Plix Extra Deep 45/45),containing five Plix fruit cut-outs to prevent fruit from moving), and40 mL SDW added to two sterile paper towels that were placed beneath thePlix cut-outs, then placed into large (40 L) plastic bins which wereclosed to ensure high relative humidity for the first 48 h and incubatedon the lab bench with natural and fluorescent light at 24° C. for up tothree days. After 48 h, the Plix lunch boxes were removed from the binsand a pin placed between the lid and base to allow air to circulate andthe relative humidity to decline over a 15 h period (5 pm in the lateafternoon to 8 am the next morning) after which, they were resealedagain. This process of alternating the relative humidity within theincubation chambers was repeated over the duration of the experiment andis a method that has been shown to avoid excessive mycelial growth.After 6 to 7 days the severity of fungal rot infection was assessed foreach treatment by measuring the lesion length (mm) along the axis of thefruit. Data were expressed as the average lesion length, minus theinitial width of the wound (3 mm).

Experimental Design

The kiwifruit ‘Hongyang’ assay 8 consisted of 4 ‘Hongyang’ kiwifruit perreplicate and there were five replicates for each treatment in arandomised block layout. In total there were 22 treatments, including aNil (no wound and no treatment) no pathogen inoculation) control, and aNil (plus wound then SDW+Tw80) no pathogen inoculation control.

In Assay 9

The kiwifruit ‘Hongyang’ assay 9 consisted of 4 ‘Hongyang’ kiwifruit perreplicate and there were four replicates for each treatment in arandomised block layout. In total there were 5 treatments, including aNil (no wound and no treatment) no pathogen inoculation control, and aNil (plus wound then SDW+Tw80) no pathogen inoculation control.

Statistical Analysis

Data were analysed using GenStat, 13th edition, with a randomised blockexperimental design and analysis of variance. Average lesion diameterdid not require data transformation to equalise the variance and rawdata means and Least Significant Differences (LSDs) are presented.

Results

Assay 8

YBCA5 treated fruit had significantly smaller lesions in ‘Hongyang’fruit than the Rovral

Aquaflo, Serenade Opti treatments and the untreated control when thefruit were inoculated with Alternaria, Colletotrichum, Penicillium andPhomopsis. YBCA5 treated fruit had lesions not significantly smallerthan Rovral Aquaflo when the fruit were inoculated with Botrytis spp.,but they were significantly smaller than the Serenade Opti treatment anduntreated control.

Rovral Aquaflo treated fruit had lesions significantly smaller than theuntreated control when fruit were inoculated with Botrytis spp. andPenicillium spp. Serenade Opti treated fruit had lesions significantlysmaller than the Untreated control when fruit were inoculated withPhomopsis (FIG. 14).

In assay 9, YBCA5 treated fruit also had significantly smaller lesionsin ‘Hongyang’ fruit than the Rovral Aquaflo, Serenade Opti treatmentsand the untreated control when the fruit were inoculated withCryptosporiopsis (FIG. 15).

Discussion

YBCA5 demonstrated activity against a range of postharvest kiwifruitfruit pathogens when it was allowed to colonise a wound site 24 hoursbefore a pathogen was introduced to the same wound. Wounding works wellas an experimental technique to demonstrate the activity of somebiopesticides.

Overall, Rovral Aquaflo performed poorly in these experiments, and thisagrichemical may be unsuitable for wound protection assays and againstthe pathogens used in this study.

Serenade Opti performed poorly in these assays, and this biopesticidemay be unsuitable as a wounded fruit wound protectant against thepathogens used in these assays.

Example 5—Yeast biocontrol of PSA in the Field SUMMARY

Grower Standard Treatment: Kocide Opti+1×Kasumin

1 Product: 2 Active Ingredient (Al) 3 Kocide: 4 Copper Hydroxide 5Kasumin: 6 Kasugamycin 7 ActiGard: 8 Acibenzolar-S-methyl

Methodology: all plants had an application of copper at bud break, thetreatment group then received the yeast treatment while the controlsreceived no treatment and grower standard respectively.

Field Trials 2015-16

Methods

Two field trial sites were established during spring 2015 with theintention of running across two consecutive seasons. The two ‘Hayward’blocks (coded Block B and C) were located in separate orchards, nearMaketu, Bay of Plenty. Block C was the same orchard as the 2014-15trial, but a different area in the blocks was used. Vines in all blockswere pergola trained with a single vine per bay. The vines generallylooked healthy at the commencement of the trial, but had suffered fromsignificant Psa symptoms 2-3 years previously, according to the grower.

The spray treatments were applied to individual vines (eight replicatesper treatment) laid out in a randomised block design. The treatmentswere:

-   -   1. Nil—no Psa control products applied during the growing season    -   2. Grower standard—copper-based foliar spray programme,        including up to one antibiotic spray    -   3. YBCA5—yeast-based foliar spray programme (2×10⁷ CFU/mL)

Spray applications in the Grower standard and YBCA5 treatments wereapplied according to the schedule in Table 5. Common agriculturaladjuvants were added to the Grower standard (0.04% Du-Wett®) and YBCA5(0.03% Nu-Film-17®) applications.

TABLE 5 Application dates in Blocks B and C for the Grower standard andYBCA5 treatment to kiwifruit against Pseudomonas syringae pv. actinidiae(Psa) from bud burst to post flowering during the 2015-16 season. BlockB Block C Date Grower Grower Treatment standard YBCA5 standard YBCA5Oct. 6, 2015 Kocide Opti¹ YBCA5² — — Oct. 7, 2015 — — Kocide Opti YBCA5Oct. 16, 2015 Kasumin³ YBCA5 Kasumin YBCA5 Oct. 27, 2015 Kocide OptiYBCA5 Kocide Opti YBCA5 Nov. 4, 2015 — — Kocide Opti YBCA5 Nov. 6, 2015Kocide Opti YBCA5 — — Nov. 19, 2015 Nil YBCA5 Nil YBCA5 (flowering)(flowering) Dec. 21, 2015 Nil (post YBCA5 Nil (post YBCA5 fruit set)fruit set) ¹Kocide ® Opti ™ applied at 70 g/100 L with Du-Wett ® (0.04%)²YBCA5 applied at 100 g/100 L with Nu-Film-17 ® (0.03%) ³Kasumin ®applied at 500 g/100 L (no adjuvant).

The YBCA5 yeast was produced by liquid fermentation, sourced from threeseparate production facilities: PFR (Ruakura), AgResearch (Lincoln) andCallaghan Innovation (Lower Hutt). A concentrated yeast pellet aftercentrifugation was supplied to the laboratory at Ruakura and this wasmixed with an inert carrier and extruded to form granules which were airdried overnight in a laminar flow hood. The number of colony formingunits/g was calculated by dissolving 0.2 g samples into 20 mL phosphatebuffered saline amended with 0.05% Tween 80 (PBSTw). This was seriallydiluted and 10-4 droplets of each dilution were transferred to Petridishes with malt yeast extract agar amended with chloramphenicol. Petridishes were incubated for 24 h at 25° C. and then 4-6° C. for a further24 h, prior to counting the number of colonies. The concentration ofYBCA5 in the granules was 2.1×10¹⁰ CFU/g. All granules were stored inairtight containers in a refrigerator (4-6° C.) and were weighed out ata rate of 100 g/100 L to achieve the target spray concentration of 2×10⁷CFU/mL.

Psa disease assessments were carried out in the ‘Hayward’ blocks justprior to flowering on 11 Nov. 2015 by visually assessing leaves for Psaspotting severity (% leaf area with necrosis). Assessments were carriedout on leaves positioned between the second and third wire out from themain cordon. Assessments were commenced after taking one step (i.e. 1 m)from the edge of the plot and a set of 25 leaves were scored. Thisprocess was repeated after taking another step down the plot. This wasthen repeated down the other side of the vine so that four sets of 25leaves (total=100) were scored within each plot. Similarly, 100buds/plot were scored for the severity of bud-browning, as describedabove.

Data were expressed as Psa incidence (based on the proportion of leaveswith Psa spotting/proportion of buds with brown sepals) and average Psaseverity (mean % leaf area necrotic/mean number of brown sepals onbuds). Data were log-transformed and analysed by ANOVA using Genstat(ver. 16) to determine treatment differences. Raw data are presentedwith statistical differences indicated based on the log-transformeddata. Analysis was also carried out by combining data from both orchardsites, after checking for any significant site x treatment interaction.

Results

Analysis of the incidence and severity of leaves with leaf spotting(necrosis), and similarly for bud symptoms, indicated that there was nosignificant Treatment x Orchard interaction; therefore data arepresented as the mean of the two orchards (Blocks B and C).

The nil control had a mean incidence of leaves with necrosis of 50% andthis was significantly reduced (P<0.05) by the Kocide Opti and two YBCA5treatments (Table 6). The efficacy of the YBCA5 was 33%. The Growerstandard treatment (Kocide Opti+Kasumin) had an efficacy of 74% andresulted in a further significant reduction in the incidence of leaveswith necrosis, compared with the YBCA5 and Kocide Opti treatments.

The mean severity of leaf spotting was only 0.24% in the nil control(Table 6). However, there was still a significant reduction (P<0.05) inthe mean severity of leaf necrosis in each of the treatments, comparedwith the nil control, including the two YBCA5 treatments. The averageefficacy of the YBCA5 was 58%, compared with the Grower standard andKocide Opti treatments which had an efficacy of 91 and 73%,respectively.

The incidence of flower buds in the nil control with necrotic sepals was61% and this was significantly (P<0.05) reduced by each of thetreatments (Table 6). The two YBCA5 treatments had 39 and 37% incidenceof buds with necrosis and an average efficacy of 38%. The GrowerStandard treatment had significantly (P<0.05) less bud necrosis (13%)compared with each of the other treatments (efficacy 79%). The KocideOpti treatment had an efficacy of 40%.

The mean severity of bud necrosis (number of necrotic sepals/bud) was1.29 in the Nil control and similarly to the incidence of buds withnecrosis, this was significantly (P<0.05) reduced by each of thetreatments, with the Grower Standard treatment (0.21, efficacy =84%)providing the biggest reduction (Table 6). The average efficacy of theYBCA5 treatments was 46% and this was similar to the Kocide Optitreatment (efficacy 38%).

TABLE 6 Mean incidence and severity of leaf spotting and bud-browning onActinidia chinensis var. deliciosa ‘Hayward’ vines in Block B and Cfollowing foliar application of YBCA5 in comparison to a Kasumin ® andKocide ® Opti ™ based Grower standard foliar treatment and Kocide Optionly, assessed on Nov. 11, 2015. Severity of % leaves Severity of % budswith bud with spotting spotting necrosis browning Treatment (incidence)(% leaf area) (incidence) (No. sepals) Nil control   50 a   0.24 a   61a   1.29 a Grower std   13 c   0.02 b   13 c   0.21 c YBCA5   33 b  0.11 b   39 b   0.68 b Kocide   27 b   0.07 b   45 b   0.80 b Opti *            YBCA5 *   34 b   0.10 b   37 b   0.72 b LSD    8.0   0.099   9.9   0.226 P value  <0.001 <0.001  <0.001 <0.001 Grower std is oneapplication Kasumin ® and three applications of Kocide ® Opti ™ appliedin Du Weft ® (0.04% v/v) YBCA5 is a formulated developmental biologicalcontrol agent applied at 100 g/100 L in Nu-Film-17 ® (0.03% v/v)Analysis performed by combining data from the two orchard trial sites *Treatment where Trichoderma was soil applied to plots 1 day prior tothese disease assessments LSD is Least significant difference (P < 0.05)Means followed by the same letters are not significantly different toeach other (P < 0.05).

Field Trials 2016-17

Methods

During the winter months the grower applied his standard winter sprayprogramme of Kocide Opti and in the spring the same treatments as abovewere applied to the same vines. Spray applications commenced with thegrower applying Kocide Opti across the trial block at early bud-burst (2Oct. 2016) and this was then followed by the schedule outlined in Table7, with the nil control plots receiving no spray applications. TheTrichoderma treatments were also continued in these two field trials.

The YBCA5 yeast granules used this season were produced as apre-commercial batch (YBCA5 e-nema-2) by the manufacturing company,e-nema GmbH, by liquid fermentation and fluidized bed drying. Granulesof YBCA5 were imported to New Zealand on 23 Sep. 2016 and were stored inan air tight container at 4-6° C. until required for treatmentapplication. These granules had a mean CFU/g of 3×10¹⁰ on 26 Sep. 2016and 2.6×10¹⁰ when tested for viability on 2 Nov. 2016.

The application rate for YBCA5 this season was set at the likelycommercial rate of 50 g/100 L (to achieve a minimum concentration of1×10⁷ CFU/mL) and was not adjusted for the actual viability in thegranules, indicating that the actual application dose ranged from1.25-1.5×10⁷ CFU/mL. Disease assessments on leaves and buds were carriedout as described for the previous season (above).

TABLE 7 Application dates in Blocks B and C for the Grower standard andYBCA5 treatment to kiwifruit against Pseudomonas syringae pv. actinidiae(Psa) from budburst to post flowering during the 2016-17 season. Block BBlock C Date Grower Grower Treatment standard YBCA5 standard YBCA5 Oct.11, 2016 Kocide Opti¹ YBCA5² Kocide Opti YBCA5 Oct. 18, 2016 Kasumin³YBCA5 Kasumin YBCA5 Oct. 27, 2016 Kocide Opti YBCA5 Kocide Opti YBCA5Nov. 8, 2016 Kocide Opti YBCA5 Kocide Opti YBCA5 Nov. 14, 2016 Nil YBCA5Nil YBCA5 (flowering) (flowering) Dec. 5, 2016 Nil (post YBCA5 Nil (postYBCA5 fruit set) fruit set) ¹Kocide ® Opti ™ applied at 70 g/100 L withDu-Wette (0.04%) ²YBCA5 applied at 50 g/100 L with Bond ® Xtra (0.06%)³Kasumin ® applied at 500 g/100 L (no adjuvant).

Psa disease assessments on flower buds were carried out in the ‘Hayward’blocks just prior to flowering on 11 Nov. 2016 by visually assessing 100buds/plot for the severity of bud-browning, as described above. Leaveswere assessed for Psa spotting severity (% leaf area with necrosis) inthese trial plots on 18 Nov. 2016. As described above, assessments werecarried out on leaves positioned between the second and third wire outfrom the main cordon. Assessments were commenced after taking one step(i.e. 1 m) from the edge of the plot and a set of 25 leaves were scored.This process was repeated after taking another step down the plot. Thiswas then repeated down the other side of the vine so that four sets of25 leaves (total=100) were scored within each plot.

Results

In the second year of this field trial the incidence of leaf spottingwas 31% in the nil control and this was significantly reduced in the twoYBCA5 treatments (Table 8). The efficacy of the YBCA only treatment was42%. The Grower standard treatment, which included the bactericideKasumin, provided a significant further reduction in the incidence ofleaf spotting, compared with the YBCA5 treatments (efficacy=74%).

The mean severity of leaf spotting was 0.19% in the nil control and thiswas significantly reduced in the YBCA5 treatments, compared with the nilcontrol (Table 8). The efficacy of the YBCA only treatment was 53%.There was a further reduction in the Grower standard treatment, but inthis case the Grower standard and YBCA5 treatments were notsignificantly different to each other.

In the nil control, the incidence of buds with necrosis was 18% and theseverity of the necrosis was 0.27. The two YBCA5 treatmentssignificantly reduced bud incidence and severity compared with the nilcontrol (efficacy of YBCA5 only=56 and 59%, respectively). Although theGrower standard had less disease than the YBCA5 treatments these werenot significantly different.

TABLE 8 Mean incidence and severity of leaf spotting and bud-browning onActinidia chinensis var. deliciosa ‘Hayward’ vines in Block B and Cfollowing foliar spray application of YBCA5 in comparison to a Kasumin ®and Kocide ® Opti ™ based Grower standard foliar treatment and aTrichoderma treatment, assessed on Nov. 11, 2016 (buds) and Nov. 18,2016 (leaves). Severity Severity % of % of leaves spotting buds bud with(% with browning spotting leaf necrosis (No. Treatment (incidence) area)(incidence) sepals) Nil control   31 a 0.19 a   18 a   0.27 a Grower std   8 c 0.02 c    4 b   0.05 b YBCA5   18 b 0.09 bc    8 b   0.11 bTrichoderma   29a 0.17 ab   14 a   0.20 a YBCA5 *   18 b 0.08 bc    8 b  0.09 b LSD    8.4 0.092    5.1   0.085 P value  <0.001 ** 0.005 <0.001 <0.001 Grower std is one application Kasumin ® and threeapplications of Kocide ® Opti ™ applied in Du Wett ® (0.04% v/v) YBCA5is a formulated developmental biological control agent applied at 100g/100 L in Bond ® Xtra (0.03% v/v) Analysis performed by combining datafrom the two orchard trial sites * YBCA5 treatment where Trichoderma hadbeen applied to the soil of these plots on three occasions during theprevious 12 months ** There was a significant site × treatmentinteraction (P = 0.016) for this variable, such that there was nosignificant treatment effects in Block C and in Block B there was ahighly significant treatment effect (P < 0.001) with the treatmentdifference being the same as indicated in this combined analysis LSD isLeast significant difference (P < 0.05) Means followed by the sameletters are not significantly different to each other (P < 0.05).

Conclusion:

YBCA5 treatment showed significant reductions in incidence and severityof leaf spotting and bud browning in ‘Hayward’ vines.

It will be appreciated that the above description is provided by way ofexample only and that variations in both the materials and techniquesused which are known to those persons skilled in the art arecontemplated.

Although the invention has been described by way of example and withreference to particular embodiments, it is to be understood thatmodifications and/or improvements may be made without departing from thescope of the invention.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognise thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

In this specification where reference has been made to patentspecifications, other external documents, or other sources ofinformation, this is generally for the purpose of providing a contextfor discussing the features of the invention. Unless specifically statedotherwise, reference to such external documents is not to be construedas an admission that such documents, or such sources of information, inany jurisdiction, are prior art, or form part of the common generalknowledge in the art.

The Following Numbered Paragraphs Define Particular Aspects of thePresent Invention:

-   -   1. Isolated Aureobasidium pullulans yeast strain YBCA5 (CBS        Accession # 141880).    -   2. A composition comprising YBCA5 and an agriculturally        acceptable carrier.    -   3. A composition consisting essentially of YBCA5 and an        agriculturally acceptable carrier.    -   4. The composition of paragraph 2 or paragraph 3, wherein YBCA5        is present in the form of reproductively viable cells.    -   5. The composition of any one of paragraphs 2 to 4, wherein the        concentration of YBCA5 viable cells ranges from about 1×10³ to        about 1×10¹⁴, preferably about 1×10⁵ to about 1×10¹¹, preferably        about 1×10⁶ to about 1×10⁹, preferably about 1×10⁷ to about        1×10⁸, preferably about 2×10⁷ to about 2×10⁸ CFU, preferably        about 2×10⁹ to about 2×10¹⁰ CFU per gram for solid compositions,        and about 1×10⁷ to about 1×10⁸ CFU per millilitre for liquid        compositions.    -   6. The composition of any one of paragraphs 2 to 5, wherein the        agriculturally acceptable carrier is water.    -   7. The composition of any one of paragraphs 2 to 6, further        comprising at least one agriculturally acceptable adjuvant.    -   8. The composition of any one of paragraphs 2 to 7, wherein the        agriculturally acceptable adjuvant is selected from the group        consisting of an additional active agent and a formulation        agent.    -   9. The composition of any one of paragraphs 2 to 7, wherein the        agriculturally acceptable adjuvant is one or more additional        active agents.    -   10. The composition of any one of paragraphs 2 to 7, wherein the        agriculturally acceptable adjuvant is one or more formulation        agents.    -   11.The composition of any one of paragraphs 2 to 7, wherein the        agriculturally acceptable adjuvant comprises combination of one        or more additional active agents and one or more formulation        agents.    -   12. The composition of any one of paragraphs 2 to 11, wherein        the composition is formulated as a solid or a liquid        formulation.    -   13. The composition of any one of paragraphs 2 to 12 wherein the        composition is a pre-prepared composition or wherein the        composition is in a concentrated form.    -   14.A method of controlling Pseudomonas spp. bacteria on a plant        or part thereof, the method comprising contacting the plant or        part thereof with YBCA5, or a composition of any one of        paragraphs 2 to 13.    -   15. The method of paragraph 14, wherein the at least one strain        of Pseudomonas spp. bacteria is a strain of bacteria selected        from the group consisting of P. syringae, P. amygdalia, P.        avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P.        ficuserectae, P. helianthi, P. lemiae, P. savastanoi, and P.        viridiflava, or a pathovar thereof, or combinations thereof,        preferably the at least one strain is P. syringae or a pathovar        thereof, more preferably the at least one strain is P. syringae        pv. actinidiae (Psa).    -   16. The method of paragraph 14 or 15, wherein the plant or part        thereof is contacted for a time sufficient to control        Pseudomonas spp. bacteria, preferably Psa bacteria.    -   17. The method of paragraph 16, wherein contacting is for a time        sufficient to reduce the survival, growth and/or proliferation        of Pseudomonas spp. bacteria, preferably Psa bacteria.    -   18. The method of any one of paragraphs 14 to 17, wherein        contacting comprises applying YBCA5, or the composition of any        one of paragraphs 2 to 13 to the plant leaves, stems, flowers,        fruits, trunks and/or roots or part thereof.    -   19.The method of paragraph 18, wherein applying comprises        dusting, spraying, dripping, sprinkling, or mixing, or        combinations thereof.    -   20. The method of paragraph 18, wherein applying to the roots is        by ground spraying, mechanical incorporation or by mixing with        enriching agents or fertilizers prior to the application of said        enriching agents or fertilizers.    -   21.The method of any one of paragraphs 14 to 20, wherein the        plant or part thereof is selected from the group of        monocotyledonous plants, dicotyledonous plants, annual, biannual        and perennial plants, vegetable plants or harvested vegetables,        fruit plants or trees or harvested fruits, flower bearing plants        or trees or harvested flowers, cereal plants, oleaginous plants,        proteinous plants, ligneous plants, and ornamental plants.    -   22.The method of any one of paragraphs 14 to 21, wherein the        plant or part thereof is an agriculturally important plant,        cultivar thereof, or product thereof selected from the group        consisting of agriculturally important vines and agriculturally        important fruit trees, and cultivars and products thereof,        preferably wherein the agriculturally important fruit trees or        cultivars thereof are selected from olive trees, apple trees,        pear trees, citrus fruit trees, banana trees, pineapple trees,        peach trees, apricot trees, cherry trees, walnut trees and        hazelnut trees and the products thereof are olives, apples,        pears, citrus fruits, bananas, pineapples, peaches, apricots,        cherries, walnuts and hazelnuts respectively, preferably wherein        the agriculturally important vines or cultivars thereof are        selected from potato vines, beetroot vines, bean vines, pea        vines, tomato vines, cucumber vines, melon vines, berry vines,        grape vines and kiwifruit vines and the products thereof are        potatoes, beetroots, beans, peas, tomatoes, cucumbers, melons,        berries, grapes and kiwifruits respectively, preferably wherein        the agriculturally important vine is a kiwifruit vine or        cultivar thereof, and the product is kiwifruit.    -   23.The method of paragraph 22, wherein the kiwifruit vine is        selected from the group consisting of species of fuzzy kiwifruit        (Actinidia deliciosa), golden kiwifruit (A. chinensis var.        chinensis), Chinese egg gooseberry (A. coriacea), baby kiwifruit        (A. arguta), Arctic kiwifruit (A. kolomikta), red kiwifruit (A.        melanandra, A. chinensis var. chinensis), silver vine (A.        polygama), and purple kiwifruit (A. purpurea) or a cultivar        thereof, preferably wherein the kiwifruit are selected from the        group consisting of A. chinensis var. deliciosa and A. chinensis        var. chinensis species or a cultivar thereof, preferably wherein        the kiwifruit is a species of A. chinensis, preferably wherein        the kiwifruit is A. chinensis var. chinensis Planch, preferably        wherein the cultivar is a ‘Hayward’ or ‘Hort16A’ variety        cultivar.    -   24. The method of paragraph 23, wherein the cultivar is A.        chinensis var. chinensis Planch, ‘Hort16A’.    -   25. The method of paragraph 23, wherein the cultivar is A.        chinensis var. deliciosa ‘Hayward’.    -   26.The method of any one of paragraphs 12 to 25, the plant or        part thereof is an agriculturally important crop plant, cultivar        or product thereof selected from corn plants, tobacco plants,        wheat plants, sugar cane plants, rapeseed plants, barley plants,        rice plants, sorghum plants, millet plants, soya bean plants,        lettuce plants, and cabbage plants.    -   27.A method for controlling P. syringae pv. actinidiae (Psa) on        a kiwifruit plant or part thereof, the method comprising        contacting the kiwifruit plant or part thereof with YBCA5, or a        composition of any one of paragraphs 2 to 13 to a species of A.        chinensis var. deliciosa or A. chinensis var. chinensis, or a        cultivar thereof, preferably a species of A. chinensis var.        chinensis, or cultivar thereof, preferably wherein the kiwifruit        plant is ‘Hort16A’ or ‘Hayward’.    -   28.A method for increasing the yield of a kiwifruit plant        infected, or susceptible to infection by Pseudomonas spp.,        preferably infected, preferably susceptible to infection with        Psa, the method comprising applying YBCA5, or a composition of        any one of paragraphs 2 to 13 to the kiwifruit plant or part        thereof.    -   29.A method of controlling at least one phytopathogenic fungus        on a plant or part thereof, the method comprising contacting the        plant or part thereof with YBCA5, or a composition of any one of        paragraphs 2 to 13.    -   30.A method for increasing the yield of a fruit or vegetable        plant infected, or susceptible to infection by a phytopathogenic        fungus, the method comprising applying YBCA5, or a composition        of any one of paragraphs 2 to 13 to the fruit or vegetable plant        or part thereof, and growing the plant or part thereof.    -   31. Use of YBCA5, or a composition of any one of paragraphs 2 to        13 for controlling Pseudomonas spp. bacteria on a plant or part        thereof.    -   32. Use of YBCA5, or a composition of any one of paragraphs 2 to        13 for controlling Psa on a kiwifruit plant or part thereof.    -   33. Use of YBCA5, or a composition of any one of paragraphs 2 to        13 for controlling a phytopathogenic fungus on a fruit or        vegetable plant or part thereof.    -   34. Use of YBCA5, or a composition of any one of paragraphs 2 to        13 for increasing the yield of a kiwifruit plant or part thereof        infected, or susceptible to infection with Psa.    -   35. Use of YBCA5, or a composition of any one of paragraphs 2 to        13 for increasing the yield of a fruit or vegetable plant or        part thereof infected with, or susceptible to infection by a        phytopathogenic fungus.    -   36. YBCA5, or a composition of any one of paragraphs 2 to 13,        for use in, or when used, for controlling Pseudomonas spp.        bacteria on a plant or part thereof.

37. YBCA5, or a composition of any one of paragraphs 2 to 13, for usein, or when used, for controlling Psa on a kiwifruit plant or partthereof.

-   -   38. YBCA5, or a composition of any one of paragraphs 2 to 13,        for use in, or when used, for controlling a phytopathogenic on a        kiwifruit plant or part thereof.    -   39. YBCA5, or a composition of any one of paragraphs 2 to 13,        for use in, or when used, for increasing the yield of a        kiwifruit plant or part thereof infected, or susceptible to        infection with Pseudomonas spp.    -   40. YBCA5, or a composition of any one of paragraphs 2 to 13,        for use in, or when used, for increasing the yield of a        kiwifruit plant or part thereof infected, or susceptible to        infection with Psa.    -   41. YBCA5, or a composition of any one of paragraphs 2 to 13,        for use in, or when used, for increasing the yield of a fruit or        vegetable plant or part thereof infected with, or susceptible to        infection by a phytopathogenic fungus.    -   42.At least one plant or part thereof treated with YBCA5, or a        composition of any one of paragraphs 2 to 13 according to a        method of any one of paragraphs 14 to 30 or according the uses        of any one of paragraphs 31 to 35.

Industrial Application

The isolated Aureobasidium pullulans yeast strain YBCA5 and compositionscomprising or consisting essentially of YBCA5 of the present inventionfind a use in controlling phytopathogenic bacteria and fungi.

1-24. (canceled)
 25. A composition comprising Aureobasidium pullulansyeast strain YBCA5 (CBS Accession # 141880) and an agriculturallyacceptable carrier.
 26. The composition of claim 25 wherein YBCA5 ispresent in the form of reproductively viable cells.
 27. The compositionof claim 25, wherein the concentration of YBCA5 viable cells ranges fromabout 1×10⁵ to about 1×10¹¹ CFU per gram for solid compositions, andabout 1×10⁷ to about 1×10⁸ CFU per millilitre for liquid compositions.28. The composition of claim 25, wherein the concentration of YBCA5viable cells ranges from about 2×10⁷ to about 2×10⁸ CFU per gram forsolid compositions, and about 1×10⁷ to about 1×10⁸ CFU per millilitrefor liquid compositions.
 29. The composition of claim 25, wherein theconcentration of YBCA5 viable cells ranges from about 2×10⁹ to about2×10¹⁰ CFU per gram for solid compositions, and about 1×10⁷ to about1×10⁸ CFU per millilitre for liquid compositions.
 30. The composition ofclaim 25, further comprising at least one agriculturally acceptableadjuvant.
 31. A method of controlling Pseudomonas spp. bacteria on aplant or part thereof, the method comprising contacting the plant orpart thereof with YBCA5, or a composition of claim
 25. 32. The method ofclaim 31, wherein the at least one strain of Pseudomonas spp. bacteriais a strain of bacteria selected from the group consisting of P.syringae, P. amygdalia, P. avellanae, P. caricapapayae, P. cichorii, P.coronafaciens, P. ficuserectae, P. helianthi, P. lemiae, P. savastanoi,and P. viridiflava, or a pathovar thereof, or combinations thereof, 33.The method of claim 31, wherein the at least one strain of Pseudomonasspp. bacteria is P. syringae or a pathovar thereof.
 34. The method ofclaim 31, wherein the at least one strain of Pseudomonas spp. bacteriais P. syringae pv. actinidiae (Psa).
 35. The method of claim 31, whereinthe plant or part thereof is an agriculturally important plant, cultivarthereof, or product thereof, selected from olive trees, apple trees,pear trees, citrus fruit trees, banana trees, pineapple trees, peachtrees, apricot trees, cherry trees, walnut trees and hazelnut trees,wherein the products thereof are olives, apples, pears, citrus fruits,bananas, pineapples, peaches, apricots, cherries, walnuts and hazelnuts,respectively.
 36. The method of claim 31, wherein the plant or partthereof is an agriculturally important plant, cultivar thereof, orproduct thereof, selected from potato vines, beetroot vines, bean vines,pea vines, tomato vines, cucumber vines, melon vines, berry vines, grapevines and kiwifruit vines, and the products thereof are potatoes,beetroots, beans, peas, tomatoes, cucumbers, melons, berries, grapes andkiwifruits, respectively.
 37. The method of claim 36, wherein theagriculturally important plant, cultivar thereof, or product thereof, isa kiwifruit vine or cultivar thereof, and the product is kiwifruit. 38.The method of claim 37, wherein the kiwifruit vine is selected from thegroup consisting of species of green-fleshed kiwifruit (Actinidiachinensis var. deliciosa), golden kiwifruit (A. chinensis var.chinensis), Chinese egg gooseberry (A. coriacea), baby kiwifruit (A.arguta), Arctic kiwifruit (A. kolomikta), red kiwifruit (A. melanandra,A. chinensis var. chinensis), silver vine (A. polygama), and purplekiwifruit (A. purpurea) or a cultivar thereof.
 39. The method of claim37, wherein the kiwifruit are selected from the group consisting of A.chinensis var. deliciosa and A. chinensis var. chinensis species or acultivar thereof.
 40. The method of claim 37, wherein the kiwifruit is a‘Hayward’, ‘Hort16A’ or ‘Hongyang’ variety cultivar.
 41. A method ofcontrolling at least one phytopathogenic fungus on a plant or partthereof, the method comprising contacting the plant or part thereof withYBCA5, or a composition of claim
 25. 42. A method of claim 41, whereinthe phytopathogenic fungus is a Monilinia spp. Fungus.
 43. The method ofclaim 41, wherein the phytopathogenic fungus is Monilinia fruticola. 44.The method of claim 41, wherein the phytopathogenic fungus is selectedfrom the group consisting of Botrytis spp., Colletotrichum spp.,Penicillium spp., Phomopsis spp., Alternaria spp., Sclerotinia spp., andCryptosporiopsis spp. fungus.